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Methods in
Molecular Biology 2300

Mathieu Rederstorff Editor

Small Non-
Coding RNAs
Methods and Protocols
Second Edition
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

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For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
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Small Non-Coding RNAs

Methods and Protocols

Second Edition

Edited by

Mathieu Rederstorff
Université de Lorraine, CNRS, IMoPA, Nancy, France
Editor
Mathieu Rederstorff
Université de Lorraine
CNRS, IMoPA
Nancy, France

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-1385-6 ISBN 978-1-0716-1386-3 (eBook)
https://doi.org/10.1007/978-1-0716-1386-3

© Springer Science+Business Media, LLC, part of Springer Nature 2015, 2021

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Preface

Small Non-Coding RNAs: Methods and Protocols is a laboratory protocols book dedicated to
biochemists or cellular/molecular biologists, already working in the field of RNA biology or
willing to start studying small non-coding RNAs in their projects.
Owing to their small sizes, tri-dimensional structures, low abundances, or differential
expression levels, small non-coding RNAs require customized dedicated protocols for their
identification and study, compared, for instance, to messenger RNAs (mRNAs).
This volume contains basic as well as more sophisticated, state-of-the-art methods to
tackle all aspects of small non-coding RNA biology, from their isolation and precipitation,
through their quantification, accurate detection, functional analysis, to their high-
throughput or even structural analysis.
This survey of technologies will be of valuable help to all the researchers interested in
studying the numerous functions of small non-coding RNAs.
I sincerely thank all the authors for their contribution to this exciting second edition.

Nancy, France Mathieu Rederstorff

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

PART I INTRODUCTION TO SMALL NON-CODING RNAS

1 Regulatory Non-Coding RNAs: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Camille Virciglio, Yoann Abel, and Mathieu Rederstorff
2 RNA Precipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Valérie Bourguignon-Igel, Yoann Abel, and Mathieu Rederstorff
3 Quantitative and Qualitative Assessment of Small RNA Preparations . . . . . . . . . . 17
Virginie Marchand, Lilia Ayadi, Valérie Bourguignon-Igel,
and Yuri Motorin
4 Non-Coding RNA Silencing in Mammalian Cells by Antisense LNA
GapmeRs Transfection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Charbel Alfeghaly, Christelle Aigueperse, Sylvain Maenner,
and Isabelle Behm-Ansmant

PART II DETECTION OF SMALL NON-CODING RNAS

5 Northern Blot Detection of Tiny RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Jana C. Wiegard, M. Amri C. Schlüter, Olga Y. Burenina,
Elena A. Kubareva, Gabriele Klug, Arnold Grünweller,
and Roland K. Hartmann
6 Stem-Loop qRT-PCR–Based Quantification of miRNAs . . . . . . . . . . . . . . . . . . . . . 59
Yoann Abel and Mathieu Rederstorff
7 Detection and Labeling of Small Non-Coding RNAs by Splinted Ligation . . . . . 65
Marlène Vayssières, Gabrielle Bourgeois, Florian Chardon, Anne-Sophie
Tillault, and Magali Blaud
8 Fluorescence In Situ Hybridization of Small Non-Coding RNAs . . . . . . . . . . . . . 73
Valentin Vautrot, Géraud Heckler, Christelle Aigueperse,
and Isabelle Behm-Ansmant

PART III FUNCTIONAL ANALYSIS OF SMALL NON-CODING RNAS

9 Using Native RIP, UV-CLIP or fCLIP to Address Protein–RNA
Interactions In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Laeya Baldini and Stéphane Labialle
10 Purification of RiboNucleoProtein Particles by MS2-MBP Affinity
Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
Aymeric Sanchez and Sylvain Maenner

vii
viii Contents

11 Study of Genome-Wide Occupancy of Long Non-Coding RNAs

Using Chromatin Isolation by RNA Purification (ChIRP) . . . . . . . . . . . . . . . . . . . 107
Charbel Alfeghaly, Isabelle Behm-Ansmant, and Sylvain Maenner
12 Gene Reporter Assays to Study miRNA Function . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Yoann Abel and Mathieu Rederstorff
13 CRISPR/Cas9 System to Knockdown MicroRNA In Vitro
and In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Bin Yi, Kristina Larter, and Yaguang Xi

PART IV HIGH-THROUGHPUT ANALYSIS OF SMALL NON-CODING RNAS

14 Microarray Analysis of Whole-Transcriptome RNAs Including

Non-Coding RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Quentin Thuillier and Isabelle Behm-Ansmant
15 Isolation, Extraction and Deep-Sequencing Analysis of Extracellular
RNAs (exRNAs) from Human Plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Virginie Marchand, Adeline Galvanin, and Yuri Motorin
16 Analysis of the RNA and Protein Complexome by Grad-seq . . . . . . . . . . . . . . . . . 183
Jens Hör and Jörg Vogel
17 μIVC-Seq: A Method for Ultrahigh-Throughput Development
and Functional Characterization of Small RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203
Farah Bouhedda, Roger Cubi, Stéphanie Baudrey,
and Michael Ryckelynck

PART V STRUCTURAL APPROACHES

18 RNA Secondary Structure Study by Chemical Probing Methods

Using DMS and CMCT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241
Fatima Alghoul, Gilbert Eriani, and Franck Martin
19 Application of NMR Spectroscopy to Determine the 3D Structure
of Small Non-Coding RNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Marie-Eve Chagot, Marc Quinternet, Xavier Manival,
and Isabelle Lebars

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Contributors

YOANN ABEL • IGMM, CNRS, Université de Montpellier, Montpellier, France

CHRISTELLE AIGUEPERSE • Université de Lorraine, CNRS, IMoPA, Nancy, France
CHARBEL ALFEGHALY • Université de Lorraine, CNRS, IMoPA, Nancy, France
FATIMA ALGHOUL • Université de Strasbourg—Institut de Biologie Moléculaire et Cellulaire,
“Architecture et Réactivité de l’ARN” CNRS UPR9002, Strasbourg, France
LILIA AYADI • Université de Lorraine, CNRS, INSERM, IBSLOR, Nancy, France;
Université de Lorraine, CNRS, IMoPA, Nancy, France
LAEYA BALDINI • Université de Lorraine, CNRS, IMoPA, Nancy, France
STÉPHANIE BAUDREY • Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN,
UPR 9002, Strasbourg, France
ISABELLE BEHM-ANSMANT • Université de Lorraine, CNRS, IMoPA, Nancy, France
MAGALI BLAUD • Laboratoire CiTCoM “Cibles Thérapeutique et Conception des Mé
dicaments”, CNRS UMR 8038, Faculté de Pharmacie, Université de Paris, Paris, France
FARAH BOUHEDDA • Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN,
UPR 9002, Strasbourg, France
GABRIELLE BOURGEOIS • Laboratoire de Biochimie, CNRS UMR 7654, Ecole Polytechnique,
Palaiseau, France
VALÉRIE BOURGUIGNON-IGEL • Université de Lorraine, CNRS, INSERM, IBSLOR, Nancy,
France; Université de Lorraine, CNRS, IMoPA, Nancy, France
OLGA Y. BURENINA • Center of Life Sciences, Skolkovo Institute of Science and Technology,
Moscow, Russia; Chemistry Department and A.N. Belozersky Institute of Physico-Chemical
Biology, M.V. Lomonosov Moscow State University, Moscow, Russia
MARIE-EVE CHAGOT • Université de Lorraine, CNRS, IMoPA, Nancy, France
FLORIAN CHARDON • ZMBE Zentrum für Molekularbuiologie der Entzündung, Münster,
Germany
ROGER CUBI • Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN, UPR
9002, Strasbourg, France
GILBERT ERIANI • Université de Strasbourg—Institut de Biologie Moléculaire et Cellulaire,
“Architecture et Réactivité de l’ARN” CNRS UPR9002, Strasbourg, France
ADELINE GALVANIN • Université de Lorraine, CNRS, IMoPA, Nancy, France
ARNOLD GRÜNWELLER • Institute of Pharmaceutical Chemistry, Philipps-University
Marburg, Marburg, Germany
ROLAND K. HARTMANN • Institute of Pharmaceutical Chemistry, Philipps-University
Marburg, Marburg, Germany
GÉRAUD HECKLER • Université de Lorraine, CNRS, IMoPA, Nancy, France
JENS HÖR • Institute of Molecular Infection Biology, University of Würzburg, Würzburg,
Germany
GABRIELE KLUG • Institute of Microbiology and Molecular Biology, Justus-Liebig-University-
Gießen, Gießen, Germany
ELENA A. KUBAREVA • Chemistry Department and A.N. Belozersky Institute of Physico-
Chemical Biology, M.V. Lomonosov Moscow State University, Moscow, Russia
STÉPHANE LABIALLE • Université de Lorraine, CNRS, IMoPA, Nancy, France

ix
x Contributors

KRISTINA LARTER • Department of Genetics and Stanley S. Scott Cancer Center, Louisiana
State University Health Sciences Center, New Orleans, LA, USA
ISABELLE LEBARS • Université de Strasbourg, CNRS, ARN UPR 9002, 2 allée Conrad
Roentgen, Strasbourg, France
SYLVAIN MAENNER • Université de Lorraine, CNRS, IMoPA, Nancy, France
XAVIER MANIVAL • Université de Lorraine, CNRS, IMoPA, Nancy, France
VIRGINIE MARCHAND • Université de Lorraine, CNRS, INSERM, IBSLOR, Nancy, France
FRANCK MARTIN • Université de Strasbourg—Institut de Biologie Moléculaire et Cellulaire,
“Architecture et Réactivité de l’ARN” CNRS UPR9002, Strasbourg, France
YURI MOTORIN • Université de Lorraine, CNRS, INSERM, IBSLOR, Nancy, France;
Université de Lorraine, CNRS, IMoPA, Nancy, France
MARC QUINTERNET • Université de Lorraine, CNRS, INSERM, IBSLOR, Nancy, France
MATHIEU REDERSTORFF • Université de Lorraine, CNRS, IMoPA, Nancy, France
MICHAEL RYCKELYNCK • Université de Strasbourg, CNRS, Architecture et Réactivité de
l’ARN, UPR 9002, Strasbourg, France
AYMERIC SANCHEZ • Université de Lorraine, CNRS, IMoPA, Nancy, France
M. AMRI C. SCHLÜTER • Institute of Pharmaceutical Chemistry, Philipps-University
Marburg, Marburg, Germany
QUENTIN THUILLIER • Université de Lorraine, CNRS, IMoPA, Nancy, France
ANNE-SOPHIE TILLAULT • Department of Chemistry and Biochemistry, Alberta RNA
Research and Training Institute, University of Lethbridge, Lethbridge, AB, Canada
VALENTIN VAUTROT • Université de Lorraine, CNRS, IMoPA, Nancy, France
MARLÈNE VAYSSIÈRES • Laboratoire CiTCoM “Cibles Thérapeutique et Conception des Mé
dicaments”, CNRS UMR 8038, Faculté de Pharmacie, Université de Paris, Paris, France
CAMILLE VIRCIGLIO • Université de Lorraine, CNRS, IMoPA, Nancy, France
JÖRG VOGEL • Institute of Molecular Infection Biology, University of Würzburg, Würzburg,
Germany; Helmholtz Institute for RNA-based Infection Research (HIRI), Helmholtz
Centre for Infection Research (HZI), Würzburg, Germany
JANA C. WIEGARD • Institute of Pharmaceutical Chemistry, Philipps-University Marburg,
Marburg, Germany
YAGUANG XI • Department of Genetics and Stanley S. Scott Cancer Center, Louisiana State
University Health Sciences Center, New Orleans, LA, USA
BIN YI • Department of Genetics and Stanley S. Scott Cancer Center, Louisiana State
University Health Sciences Center, New Orleans, LA, USA
 Part I

Introduction to Small Non-Coding RNAs

 Chapter 1

Regulatory Non-Coding RNAs: An Overview

Camille Virciglio, Yoann Abel, and Mathieu Rederstorff

Abstract
The discovery of new classes of non-coding RNAs has always been preceded or accompanied by techno-
logical breakthroughs, and these outstanding progresses in transcriptomics approaches enabled to regularly
add new members to the list. From the first detection of tRNAs, through the revolution of miRNAs
discovery, to the recent identification of eRNAs or the identification of new functions for already known
ncRNAs, this introductive review provides a very concise historical and functional overview of most
prominent small regulatory non-coding RNA families.

Key words miRNA, snoRNA, piRNA, tRNA, rRNA, tiRNA, snRN, eRNA, ceRNA, circRNA

1 Small Non-Coding RNAs Begin

It has been more than 60 years that the first soluble cytoplasmic
small non-coding RNA (sncRNA), a yeast alanine transfer RNA
(tRNA), was identified and its primary structure determined
[1, 2]. Together with ribosomal RNAs (rRNA), the nucleic acids
moieties of the ribosomes, tRNAs play a central role in protein
synthesis, as the adaptor molecules providing the correct amino
acid in response to a specific codon on the mRNA [3]. Only
20 years later were the first nuclear RNAs uncovered: uridine-rich
RNAs, or U-RNAs, were observed to be very abundant, nucleus, or
sometimes even nucleolus specific and highly conserved [4, 5];
small nuclear RNAs (snRNA) U1, U2, U4, U5, and U6 were
later shown to be involved in splicing, in association with several
proteins within a ribonucleoprotein particle (RNP) called spliceo-
some [6]. Base pairings among snRNAs and between snRNAs and
pre-messenger RNA (pre-mRNA) regions account for the proper
conformation of both spliceosome and pre-mRNA, eventually lead-
ing to release of the introns and mature mRNAs [7]. Interestingly,
most small nucleolar RNAs (snoRNAs) are processed from the
released introns [8]. Within snoRNPs, snoRNAs guide post-
transcriptional RNA modifications of other ncRNAs, e.g., rRNAs

Mathieu Rederstorff (ed.), Small Non-Coding RNAs: Methods and Protocols, Methods in Molecular Biology, vol. 2300,
https://doi.org/10.1007/978-1-0716-1386-3_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021

3
4 Camille Virciglio et al.

and snRNAs: 20 -O-methylations for C/D boxes snoRNAs and

pseudouridylations for H/ACA boxes snoRNAs. Some snoRNAs,
such as U3 or U8, are instead involved in early cleavages of
pre-rRNA during ribosome biogenesis in the nucleolus [9–11].
In the late 1990s, pioneering studies aimed at identifying novel
non-coding RNAs using experimental approaches combining
library generation and sequencing identified numerous novel snoR-
NAs in various models including human, using material that was
usually discarded: small total RNA in the size range of 50 to
500 nucleotides [12]. At that time, looking for shorter RNAs was
considered a nonsense, and they were therefore disregarded. Non-
sense? Antisense actually!

2 miRNAs in the Spotlight

In the early 1990s, the lin-14 gene was shown to be involved in

temporal development in the worm C. elegans. The lin-4 gene
product, most probably a polypeptide, was shown to negatively
regulate lin-14 expression by directly acting on the 30 untranslated
region of its pre-mRNA [13]. In 1993, both Ambros and Ruvkun
labs discovered that the lin-4 gene product actually was a small
non-coding RNA, of 22 nucleotides in length in its mature form,
targeting the 30 UTR of the lin-14 pre-mRNA several times, owing
to imperfect antisense base pairing [14, 15]. Unfortunately
enough, apparently, the lin-4 small RNA neither had additional
counterparts in worm nor was it found to be conserved in other
species. However, in 2000, Pasquinelli and coworkers identified
that let-7, another 22 nucleotides long small temporal RNA
(stRNA) involved in C. elegans developmental timing, was con-
served in human and drosophila, anticipating the likely discovery
of many other small regulatory RNAs [16]. Revolution came less
than 1 year later, when Tuschl, Bartel, and Ambros labs reported, in
the same issue of Science, the observation of an abundant class of
novel small regulatory RNA in worm, human, and drosophila,
termed since microRNAs (miRNAs) [17–19]. Even certain viruses
were next shown to encode their own miRNAs [20, 21].
It was rapidly observed that the miRNA maturation and assem-
bly machinery was the same as the one implicated in the formation
of short interfering RNAs (siRNAs), involved in the RNA interfer-
ence (RNAi) pathway described in worm a couple of years earlier by
the 2006 Nobel Prize awardees Andrew Fire and Craig Mello
[22, 23], and even earlier in plants [24]. miRNA/siRNA matura-
tion, from a double-stranded precursor, is now well understood
[25], but still open to surprises. It was, for example, discovered that
miRNAs could be processed from other types of ncRNAs, such as
snoRNAs [11, 26, 27], with possible novel functions still to be
deciphered.
 ncRNAs Overview 5

In human, siRNAs, within the RNA-induced silencing complex

(RISC), mediate perfectly complementary mRNA target cleavages
[28, 29]. On the other hand, miRNAs regulate gene expression by
imperfectly base-pairing to the 30 UTR of the targeted mRNA,
repressing its translation, which eventually leads to the mRNA
decay by different possible mechanisms [30, 31].
However, another degree of complexity has emerged recently,
as miRNA-dependent gene expression regulation was shown to be
modulated by so-called RNA “sponges” [32], also referred to as
competitive endogenous RNAs (ceRNAs) [33, 34]. Many ceRNAs
compete with miRNA-binding sites and microRNA recognition
elements (MREs). They sequester miRNAs, which creates networks
of shared miRNAs and enables modulation of miRNA-dependent
gene expression regulation [33].
The family of circular RNAs, or circRNAs, already observed in
the early 1990s [35, 36], recently regained more interest as possible
ceRNAs members [37–39]. Their formation is generally consecu-
tive to the alternative splicing of a pre-mRNA, in a process called
“back-splicing.” In this process, the upstream splice acceptor is
connected to a downstream splice donor [40, 41]. Other functions
have been proposed for circRNAs, one of which leading to the idea
that circRNAs might also act as RNA-binding proteins “sponges”
[42] or even compete with the mRNA splicing itself [43].

3 Small Non-Coding RNAs Return

Another sncRNA family, the Piwi interacting RNA (piRNAs), dis-

covered about half a decade after miRNAs, also regulate transcrip-
tion [44]. piRNAs are 27–29 nucleotides long RNAs. They prevent
transposons dissemination in germinal cell lines owing to an origi-
nal “ping-pong” mechanism [45, 46]. The smallest ncRNAs
known to date, the transcription initiation RNAs (tiRNAs), sized
17–18 nucleotides, are generated upon stalling or backtracking of
RNA polymerase II (RNAPII) near the transcription start sites
(TSSs), and might be involved in transcription initiation regulation
as well [47, 48]. TSSs appear to be the source of bunches of small
and long non-coding RNAs (lncRNAs) [49], such as transcription
start site-associated RNAs (TSSa-RNAs) and promoter-associated
RNAs (PASRs) in animals [50], or promoter upstream transcripts
(PROMPTs) [51] and cryptic/Xrn1-sensitive unstable transcripts
(CUTs/XUTs) in yeast [52–54]. While lncRNAs are generally
involved in epigenetic and chromatin modifications [55–57], func-
tionality of short transcripts is debated. They might be involved in
maintaining chromatin into a transcriptionally active state or in
maintaining RNAPII available near TSSs [58].
6 Camille Virciglio et al.

In this respect, the recently described enhancer RNAs (eRNAs)

may feature some common functions with TSS-associated RNAs.
Enhancer elements are generally short DNA sequences promoting
transcription and were initially considered not to be transcribed
themselves, until recently [59, 60]. eRNAs’ biogenesis was since
shown to be initiated by the recruitment of activators and transcrip-
tion factors normally required for the transcription of surrounding
genes. Next, histone reorganization at the enhancer site is activated
by epigenetic modifications, which increase accessibility of the
enhancer sequence, triggering initiation of transcription, elonga-
tion, and processing of eRNAs [61]. In terms of functions, it was
demonstrated that eRNAs altered chromatin architecture and epi-
genetic modifications and could recruit proteins to the enhancers
regions [61]. In addition, transcription of eRNAs, rather than
eRNAs themselves, was shown to attenuate and fine-tune transcrip-
tion of surrounding mRNA by interfering with or pausing the
transcription machinery [62, 63].

Acknowledgments

This work was supported by the Centre National pour la Recherche

Scientifique, the Université de Lorraine and the Région Lorraine.

References

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88:745–751 8. Matera AG, Terns RM, Terns MP (2007)
2. Holley RW, Apgar J, Everett GA, Madison JT, Non-coding RNAs: lessons from the small
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 Chapter 2

RNA Precipitation
Valérie Bourguignon-Igel, Yoann Abel, and Mathieu Rederstorff

Abstract
Precipitation is a critical step to recover RNA of high purity. This chapter describes the principles of
alcoholic precipitation as well as a standard, basic protocol with key advices to observe, but numerous
variations on the theme are discussed. Indeed, several important parameters, such as the choice of salt,
alcohol, or carrier, have to be considered to improve the efficiency of precipitation and the yield of RNA
recovery.

Key words RNA, Precipitation, Alcohol, Salt, Carrier

1 Introduction

Alcoholic precipitation is used to recover, purify, or concentrate

nucleic acids from a total extract. It relies on nucleic acids differen-
tial solubility in solution upon addition of alcohol and salts. Nucleic
acids are soluble in water because both nucleic acids and water are
polar molecules that can therefore interact together [1]. The nega-
tively charged phosphate groups (PO3 ) of nucleic acids electro-
statically interact with water. Hence, water molecules form a
solvation or hydration shell around each nucleic acid molecule,
isolating them from each other and subsequently dissolving them
in solution. Alcohol and salt enable to disrupt this hydration shell.
In water, salts used for nucleic acids precipitation completely disso-
ciate into their constituting anions and cations. Cations neutralize
the negative charges of nucleic acids (PO3 groups) while alcohol,
which has a much lower dielectric constant than water, increases the
electrostatic interaction force between the phosphate groups and
the cations. Once neutralized, nucleic acids become less hydrophilic
and thus precipitate out of solution. Additionally, alcohol decreases
the repulsive forces between the inter-helical phosphates so that
nucleic acids molecules aggregate.
Depending on the sample type or the subsequent experiments
to be performed, several parameters such as the type of salt or

Mathieu Rederstorff (ed.), Small Non-Coding RNAs: Methods and Protocols, Methods in Molecular Biology, vol. 2300,
https://doi.org/10.1007/978-1-0716-1386-3_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021

11
12 Valérie Bourguignon-Igel et al.

alcohol to be used, the addition of a carrier, the precipitation

temperature, or the centrifugation speed have to be considered to
improve RNA quality and recovery yield (see Notes 1–5).

2 Materials

1. 100% ethanol.
2. 70% ethanol.
3. 100% isopropanol.
4. 3 M sodium acetate, pH 5.2.
5. 5 M ammonium acetate.
6. 8 M lithium chloride.
7. 2 M sodium chloride.
8. Yeast tRNA (10–20 μg/ml).
9. Salmon sperm DNA (10–20 μg/ml).
10. Glycogen (50–150 μg/ml).
11. GlycoBlue™ Co-precipitant (50–150 μg/ml).
12. Linear polyacrylamide (10–20 μg/ml).
13. 1 M MgCl2.
14. RNase-free, DEPC-treated water.
15. 1  TE buffer, pH 8.0: 10 mM Tris–HCl, pH 8.0,
1 mM EDTA.

3 Methods

To avoid RNase contaminations when preparing and handling

RNAs, always use appropriate precautions such as RNase-free
water (see Note 6), gloves, RNase-free tubes and tips.

3.1 Ethanol 1. Adjust sample volume to a minimum of 100 μl (see Note 7).
Precipitation 2. Add sodium acetate to the sample to a final concentration of
0.3 M (about 1/10 of volume) (see Notes 1, 3 and 8).
3. Add 2.5 to 3 volumes of ice-cold 100% ethanol (see Note 2).
4. Mix thoroughly by inverting the tube or pipetting up and
down (see Note 9).
5. Incubate on ice for 15 min to 1 h (see Note 4).
6. Centrifuge at 12,000  g for 30 min at 4  C (see Note 5).
7. Carefully discard the supernatant without disturbing the pellet.
8. Wash the RNA pellet with 0.5 ml of 70% ethanol and mix
gently.
 RNA Precipitation 13

9. Centrifuge at 12,000  g for 15 min at 4  C.

10. Carefully discard the supernatant without disturbing the pellet
(see Note 10).
11. Air-dry the RNA pellet for 5–10 min at room temperature (see
Note 11).
12. Dissolve the pellet in RNase-free water (see Note 12).

4 Notes

1. Different salts can be used during alcoholic precipitation, the

choice of which depending on the subsequent applications.
Indeed, each salt features specific properties providing different
advantages and/or drawbacks [1, 2]. Sodium acetate (0.3 M
final concentration, pH 5.2) is the most widely used salt for
alcoholic precipitation [1, 2]. However, if the sample contains
SDS, sodium chloride (0.2 M final concentration) could be
alternatively chosen. In this case, SDS would remain soluble
in ethanol [1, 2], which would allow its separation and removal
from the RNA. Ammonium acetate (2.5 M final concentration)
is another possible choice. It allows to greatly reduce
co-precipitation of both dNTPs and oligosaccharides. How-
ever, ammonium acetate should not be used if a phosphoryla-
tion reaction is planned after precipitation since ammonium
ions inhibit T4 polynucleotide kinase [1, 2]. Finally, a last
broadly used salt for RNA precipitation is lithium chloride
(0.8 M final concentration). It is very soluble in alcohol and
therefore co-precipitates with RNA to a smaller extent than
other salts [1, 2]. Moreover, DNA, proteins, and carbohydrates
do not efficiently precipitate with lithium chloride, which
therefore leads to a greater purity of RNAs precipitated with
this salt [3]. Unfortunately, possible loss of small RNAs have
been reported (100 nt or less, e.g., miRNAs, tRNAs, sn- and
snoRNAs or 5S/5.8S rRNAs). Anyway, as small and large
RNAs feature different solubility in high ionic strength solu-
tions, small RNAs remain soluble while larger ones do not. This
property enables to selectively purify small RNAs with high
concentrations of LiCl (up to 2.5 M) even without alcohol
[1], as small RNAs will remain in solution. However, lithium
chloride should not be employed if subsequent reverse tran-
scription or in vitro translation reactions are planned, as the
chloride ions at this concentration inhibit RNA-dependent
DNA polymerase as well as initiation of protein synthesis in
most cell-free systems [1].
2. Alternatively, isopropanol can replace ethanol as a precipitation
alcohol. Indeed, since isopropanol is less polar than ethanol,
14 Valérie Bourguignon-Igel et al.

RNAs are even less soluble in isopropanol than they are in

ethanol. Therefore, for a similar precipitation efficiency, a
lower volume of isopropanol is necessary [1, 2]. Isopropanol
can therefore replace ethanol in case of large sample volume,
using about one volume of isopropanol per volume of sample.
However, as salts are also less soluble in isopropanol, they
coprecipitate and contaminate RNA preparation to a higher
extent [2]. Therefore, washing steps after precipitation with
isopropanol are very important. Finally, isopropanol is less
volatile than ethanol and will require more time for the RNA
pellet to dry [2].
3. If you expect RNA quantity to be very low or if RNAs are very
small in size (<100 nucleotides), you can add a carrier or
co-precipitant to increase recovery yield before incubation on
ice. Also, if you do not see any pellet after the centrifugation
step, it is possible to proceed again to precipitation (starting
from step 5, Subheading 3.1) after addition of a carrier. Car-
riers are molecules that trigger precipitation of RNA as they are
insoluble in alcohol and thus precipitate. After centrifugation,
the pellet will consequently be bigger and thus more visible,
which will greatly facilitate removal of the supernatant without
affecting the pellet. Moreover, colored dyes can be covalently
linked to carriers, which additionally facilitates the observation
of the pellet.
The choice of the carrier in alcoholic precipitation is impor-
tant. As for the choice of the salt to be used, carrier choice will
depend on the subsequent reactions to be performed [2]. The
most frequently used carrier molecules are yeast tRNAs (final
concentration of 10–20 μg/ml), glycogen (final concentration
of 50–150 μg/ml), or linear polyacrylamide (final concentra-
tion of 10–20 μg/ml). Yeast tRNAs are biologically active
RNAs that might interfere with some subsequent current
molecular biology reactions. For example, tailing reactions or
reactions catalyzed by polynucleotide kinases or terminal trans-
ferases are inhibited by large amounts of yeast tRNAs [4]. In
some cases, template-dependant cDNA synthesis is inhibited as
well [5]. On the other hand, glycogen is an inert, DNA/RNA-
free molecule. It interferes neither with subsequent enzymatic
reactions (PCR, digestion, ligation, reverse transcription, label-
ing, transcription, hybridization, etc.) nor with electrophoresis
or spectrophotometrical measurements [6]. Glycogen even
allows improved precipitation of very small RNA (>8 nt).
However, glycogen may prevent reverse transcription in the
case of very large templates, as well as nucleic acids interactions
with proteins [7] in a concentration-dependent manner
[8]. Finally, linear polyacrylamide is another DNA/RNA-free
inert molecule. As glycogen, it does not interfere neither with
 RNA Precipitation 15

enzymatic reactions such as phosphorylation by polynucleotide

kinase, ligation, cDNA synthesis, in vitro transcription, diges-
tion by endonucleases nor with electrophoresis [9]. Linear
polyacrylamide does not inhibit nucleic acids interactions
with proteins [10]; however, it does not trigger
co-precipitation of very small RNAs (20 bases) [7].
4. The incubation step for the alcoholic precipitation of RNAs is
generally performed at very low temperature ( 20  C
or 80  C) in most protocols. However, although possible
RNases might be less active at lower temperatures, this appears
to be rather counter-productive [1, 2]. Indeed, the lower the
temperature, the higher the viscosity, which decreases RNAs
movements and thus aggregation and precipitation. Addition-
ally, the dielectric constant increases when the temperature
diminishes, therefore precipitation efficiency decreases with
the temperature. Finally, salts solubility decreases with the
temperature as well, which leads to stronger coprecipitation
of salts at lower temperatures [11]. Therefore, incubation at
room temperature or on ice is sufficient and recommended,
providing you work in RNase-free conditions. If you retrieve a
low amount of RNA or if you do not see the pellet, you can
increase the incubation time or add a carrier if not done already.
An average time of about 15 min is generally sufficient.
5. If the amount of RNA is low, if you do not see the pellet, or if
you work with very small RNAs (<100 nucleotides), you can
both increase the speed and time of centrifugation in order to
allow the pellet to more firmly stick to the tube wall [1, 2].
6. Water can be treated with DEPC (diethylpyrocarbonate) to
avoid RNases contaminations.
7. Too small volumes of samples reduce the yield of RNA
recovery.
8. MgCl2 should be added to a final concentration of 10 mM for
small RNAs (<100 nucleotides) [1].
9. Do not vortex. Too vigorous mixing could damage and slice
the RNA.
10. Great care must be taken when discarding the supernatant in
order not to lose the RNA pellet.
11. Do not dry the pellet too much, especially employing a speed-
vacuum, as too dry RNA pellets are more difficult to
resolubilize.
12. You can dissolve the pellet in 1  TE buffer, pH 8, instead of
water. Although EDTA chelates divalent cations, thus inhibit-
ing metalloenzymes such as nucleases or proteases, it might
also inhibit polymerases or other enzymes necessary for your
subsequent experiences.
16 Valérie Bourguignon-Igel et al.

References
1. Sambrook J, Fritsch EF, Maniatis T (1989) 6. Tracy S (1981) Improved rapid methodology
Molecular cloning, a laboratory manual, 2nd for the isolation oh nucleic acids from agarose
edn. Cold Spring Harbor Laboratory Press, gels. Prep Biochem 11:251–268
Cold Spring Harbor, NY, pp E.10–E.15 7. Gaillard C, Strauss F (1990) Ethanol precipita-
2. Zumbo P (2012) Ethanol precipitation. tion of DNA with linear polyacrylamide as car-
Department of Physiology & Biophysics, rier. Nucleic Acids Res 18:378
Weill Cornell Medical College 8. Baugh LR et al (2001) Quantitative analysis of
3. Barlow J et al (2001) A simple method for the mRNA amplification by in vitro transcription.
quantitative isolation of Undegraded high Nucleic Acids Res 29:E29
molecular weight ribonucleic acid. Biochem 9. Aruffo A, Seed B (1987) Molecular cloning of
Biophys Res Commun 13:61–66 a CD28 cDNA by a high-efficiency COS cell
4. Michelson AM, Orkin SH (1982) Characteri- expression system. Proc Natl Acad Sci
zation of the Homopolymer tailing reaction 84:8573–8577
catalyzed by terminal deoxynucleotidyl trans- 10. Strauss F, Varshavsky A (1984) A protein binds
ferase. J Biol Chem 257:14773–14782 to a satellite DNA repeat at three specific sites
5. Wang QT et al (2002) Yeast tRNA as carrier in that would be brought into mutual proximity
the isolation of microscale RNA for global by DNA folding in the nucleosome. Cell
amplification and expression profiling. Bio- 37:889–901
Techniques 33:788–796 11. Zeugin J, Hartley JL (1985) Ethanol precipita-
tion of DNA. Focus 7:1–2
 Chapter 3

Quantitative and Qualitative Assessment of Small RNA

Preparations
Virginie Marchand, Lilia Ayadi, Valérie Bourguignon-Igel,
and Yuri Motorin

Abstract
Recent advances in high-throughput sequencing have shed new light on the diversity of small noncoding
RNA (sncRNA) classes and their crucial roles in gene regulation and disease. One key step in sncRNA
profiling consists in their quantification and assessment of their degradation extent. In this chapter, we will
describe different gold standard methods used to achieve both purposes before using the sncRNAs in
downstream applications.

Key words RNA quantification, UV spectroscopy, Fluorescence, Capillary electrophoresis, RNA

integrity number

1 Introduction

The small noncoding RNA (sncRNAs) family is composed of dif-

ferent RNA subclasses including tRNAs and tRNA-derived frag-
ments (tRFs), miRNAs, piRNAs, snRNAS, snoRNAs, and
scaRNAs. High-throughput sequencing methods have revealed
extreme diversity of sncRNAs, many of which being initially con-
sidered as simple degradation products. Hence, RNA quantifica-
tion is an important and necessary step prior to any further analysis.
The most commonly used technique for measuring the concentra-
tion of nucleic acid samples is the determination of their absorbance
at 260 nm (A260). While this is a very fast and cheap method to
estimate RNA quantity and its relative purity, this technique reveals
to be rather inaccurate if the sncRNA sample is contaminated by
DNA, proteins, or other products such as rNTPs, carbohydrates,
phenol, DTT, or chaotropic salts, which compromise accuracy of
the optical density readings. To avoid these problems, other meth-
ods, such as fluorometry, have been developed. These methods
employ specific fluorescent dyes to selectively quantify a

Mathieu Rederstorff (ed.), Small Non-Coding RNAs: Methods and Protocols, Methods in Molecular Biology, vol. 2300,
https://doi.org/10.1007/978-1-0716-1386-3_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021

17
18 Virginie Marchand et al.

biomolecule of interest (RNA, DNA, or proteins). As these dyes

only emit signals when bound to their specific target molecule, even
at low concentrations, this enables, for instance, to distinguish
between RNA and DNA present in the same sample. Use of fluo-
rescence for RNA quantification is very robust, easy, and rapid,
moreover it tolerates various buffer compositions, even detergent-
containing buffers (see Note 1). The contamination of sncRNA
samples by proteins and/or DNA can also be measured using
protein- and/or DNA-specific kits. However, contamination by
other substances, such as phenol, cannot be detected this way.
Next, RNA quality can be assessed by two additional
approaches: conventional denaturing/non-denaturing agarose gel
electrophoresis or different versions of high-resolution capillary
electrophoresis.
Since most sncRNAs are only present in low amounts in total
RNA preparations, their integrity is difficult to evaluate directly. In
addition, length of sncRNAs may considerably vary. Thus, evalua-
tion of sncRNA, as well as mRNAs, quality in total RNA samples is
deduced from global integrity of abundant RNA species, mostly
rRNAs and, possibly, tRNAs.
Rapid assessment of RNA quality can easily be obtained by
running a sample on denaturing agarose gel [1] and inspecting
the 28S/18S rRNA ratio, but this technique requires tremendous
amount of input RNA and does not provide quantitative results. In
addition, the use of toxic and/or carcinogenic denaturing agents
such as formaldehyde, formamide, or urea has led to the develop-
ment of alternative methods, such as native agarose gels supple-
mented with sodium hypochlorite, sometimes referred to as bleach
agarose gels [2] (see Note 2).
To overcome these limitations, microfluidic-based technolo-
gies have been developed to quantify and analyze RNA integrity
in an unbiased way. Instruments such as the Agilent 2100 Bioana-
lyzer, 2200 TapeStation (Agilent Technologies), or the Experion
(Bio-Rad) are considered the gold standards for measuring RNA
quality at the picogram scale. The 28S/18S ratio is automatically
calculated and, when comprised between 1.8 and 2.0, indicates
good RNA integrity. In addition, an integrated algorithm assigns
an RNA Integrity Number (RIN, Agilent Technologies) from 1 to
10, where 10 represents a completely intact RNA and 1 a highly
degraded one [3, 4]. However, running of microfluidic chips is
relatively expensive, and some technical limitations exist as well.
Calculation of RIN is meaningful only for total RNA samples,
containing major peaks of rRNA. If the RNA preparation does
not contain rRNAs, RIN is erroneous or not calculated at all,
making it impossible to conclude on the quality of the RNA prepa-
ration. Another problem concerns RNA preparations of insect or
plant origin, as the profile for total RNA differs substantially from
other common eukaryotic sources. In plant leaves, total RNA
 Quantity and Quality Control of Small RNAs 19

contains a high proportion of rRNA species from chloroplasts (16S,

23S, and 5S rRNAs) [5], while in insects, e.g., Drosophila, the 28S
rRNA is processed in two pieces that migrate around the 18S upon
heat denaturation [6] (Fig. 2a).
This chapter focuses on the most common methods to quantify
and estimate the quality of an RNA preparation and provides several
tips to optimize these methods in order to get accurate and repro-
ducible results.

2 Materials

Prepare all solutions using RNase-free water. Wear gloves to pre-

vent degradation of RNA samples by RNases.

2.1 RNA 1. UV-visible small volume spectrophotometer: Any kind of

Quantification UV-visible spectrophotometer allowing measurements of 1 μl
samples (e.g., NanoDrop One (see Note 3) or One-C, Nano-
2.1.1 Using
Drop 2000, 2000c, or 8000, Thermo Fischer Scientific). We
a Spectrophotometer
use a NanoDrop One.
2. 1.5 ml RNase-free microcentrifuge tubes.
3. RNase-free water.

2.1.2 Using 1. Any kind of fluorometer able to quantify RNA, DNA, and
a Fluorometer proteins with high sensitivity, e.g., Qubit 3.0 Fluorometer
(Life technologies) which is the one we use.
2. Thin-walled polypropylene tubes of 500 μl, compatible with
the fluorometer (e.g., Qubit Assay Tube or Axygen PCR-05-C
tubes, VWR)
3. RNA assay kit: Qubit RNA Assay Kit (5–100 ng), Qubit RNA
BR Assay Kit (20–1000 ng), Qubit microRNA Assay Kit
(1–500 ng).
4. Optional: Qubit dsDNA HS Assay Kit (0.2–100 ng) and Qubit
Protein Assay Kit (0.25–5 μg).

2.2 RNA Quality 1. Agarose.

Assessment 2. 10 TBE buffer: 108 mg/ml Tris–HCl, pH 8, 55 mg/ml
2.2.1 Native Agarose Gel boric acid, 9.3 mg/ml EDTA, pH 8.
Electrophoresis 3. Agarose gel electrophoresis chamber.
4. Ethidium bromide (EtBr) or equivalent (e.g., GelRed, FluoP-
robes, or SYBR Safe DNA Gel Stain, Invitrogen).
5. 10x DNA loading buffer: 1.9 mM xylene cyanol, 1.5 mM
bromophenol blue, 25% glycerol.
20 Virginie Marchand et al.

2.2.2 Capillary 1. Agilent 2100 Bioanalyzer (Agilent Technologies) or Experion

Electrophoresis (Bio-Rad). Since we use an Agilent 2100 Bioanalyzer, this
chapter will focus on this machine.
2. Agilent RNA 6000 Nano Kit (25 to 500 ng/μl), Agilent RNA
6000 Pico kit (50 to 5000 pg/μl), or Agilent Small RNA kit
(50 to 2000 pg/μl) (Agilent) (Table 1).
3. Chip priming station (Agilent Technologies).
4. Heating block.
5. RNase-free water.
6. RNase-free 1.5 ml microcentrifuge tubes.

3 Methods

3.1 RNA Carry out all procedures at room temperature unless otherwise
Quantification specified.

3.1.1 Using 1. Select the “Nucleic Acids” tab from the home screen of the
a Spectrophotometer NanoDrop One spectrophotometer.
2. Pipette and load 1 μl of a blank solution (e.g., RNase-free water
or TE buffer) onto the lower pedestal and lower the arm.
3. If “Auto-blank” is On, the blank measurement starts automat-
ically, otherwise select “Blank” and wait for the measurement
to complete.
4. Lift the arm and clean both pedestals with a clean wipe.
5. Pipette and load 1 μl of the sample solution onto the pedestal,
lower the arm (see Note 4).
6. If “Auto-Measure” is On, the sample measurement starts auto-
matically, otherwise select “Measure” and wait for the measure-
ment to complete. The spectrum and reported values are
displayed.
7. When you are done with sample measurements, select “End
Experiment” and export data on a USB stick.
8. Lift the arm and clean both pedestals with a new wipe.
9. Analyze the data obtained for your different RNA samples. The
concentration of your RNA sample based on the absorbance at
260 nm is given in “ng/μl” by default setting. The “A260/
A280” ratio is provided. This ratio is used to roughly assess the
purity of your RNA preparation. A ratio of 2 is generally
considered as “pure” RNA. The “A260/A230” ratio is
provided as well. This ratio is used to assess RNA purity. For
“pure” RNAs, it should be in the range of 1.8–2.2 (see Note 5).
In addition, if you use the NanoDrop One, its technology
will enable to identify most contaminants (phenol, proteins,
etc.).
 Quantity and Quality Control of Small RNAs 21

Table 1
Overview of the technical specifications for the Agilent RNA 6000 Nano, RNA 6000 Pico, and Small
RNA kits

Agilent RNA 6000 Pico Agilent Small RNA

Agilent RNA 6000 Nano kit kit kit
Reagents and consumables included in the kit (stable for 4 months)
Number of chips/kit 25 chips
Electrode cleaner 3 chips
chips
RNA dye concentrate 1 vial (blue cap)
RNA marker 4 vials (green cap)
RNA conditioning N.A 1 vial (white cap)
solution
Gel matrix 2 vials (red cap)
Spin filters 4 spin filters 2 spin filters
Ladder 1 vial (yellow cap)
Safe-lock Eppendorf 30
PCR tubes
Syringe 1
Technical specifications
Samples analyzed/ 12 11
chip
Analysis range (nt) 20–6000 nt 6–150 nt
Sample volume 1
Quantitative range 25–500 ng/μL (5–500 ng/μ 0.2–5 ng/μl (0.05–5 ng/ 0.05–2 ng/μl for
(qualitative range) L) for total RNA μl) for total RNA purified miRNA
25–250 ng/μL (25–250 ng/μ 0.5–5 ng/μl (0.25–5 ng/
L) for mRNA μl) for mRNA
Reproducibility of 10% CV 20% CV
quantitation
Quantitation accuracy 20% CV (for ladder as sample) 30% CV (for ladder as 25% CV (for ladder
sample) as sample)
Buffer compatibility 100 mM Tris or 125 mM 50 mM Tris or 50 mM 10 mM Tris and
NaCl or 15 mM MgCl2 NaCl 0.1 mM EDTA
Analysis run time 30
(min)
22 Virginie Marchand et al.

3.1.2 Using 1. Equilibrate all solutions of the appropriate kit at room temper-
a Fluorometer ature for at least 30 min before starting the experiments. The
kit provides the concentrated assay reagent, dilution buffer,
and pre-diluted standards (see Note 6).
2. Prepare the dye working solution by diluting the concentrated
assay reagent 200 times in dilution buffer. Prepare 200 μl of
working solution for each sample and the two standards.
3. Prepare the two standards annotated “C” and “D” by mixing
10 μl of standard with 190 μl of working solution.
4. Add working solution up to 200 μl to 1–20 μl of RNA sample.
5. Vortex the tubes for 2 s and incubate them for 2 min at room
temperature.
6. On the home screen of the Qubit 3.0 Fluorometer, select the
quantification assay “RNA” for which you want to perform a
new calibration.
7. Select “Read standards” to perform a calibration.
8. When indicated, insert the standard #1 tube and select “Read
standard.” Standard #1 and #2 correspond to standards “C”
and “D,” respectively.
9. Once the calibration is done, select “Run samples” to make the
measurements.
10. Indicate with the + and buttons on the wheel, the sample
volume added to the assay tube and select the units for the
output sample concentration.
11. Insert a sample tube into the sample chamber, close the lid and
then select “Read tube.”
12. Read the results, remove the current sample and insert a
new one.
13. To address if your RNA sample preparation is contaminated by
DNA or proteins, repeat the experiment with the DNA and/or
Protein kits (see Note 7).

3.2 RNA Quality 1. Pour a 1% w/v agarose gel in 1 TBE.

Assessment 2. Heat at least 1 μg of each RNA samples for 2 min at 70  C and
3.2.1 Native Agarose Gel cool down on ice.
Electrophoresis 3. Mix with 10 DNA loading buffer.
4. Load an RNA ladder or an RNA control on the gel to estimate
RNA quantity and quality.
5. Run the gel at 10 V/cm in 1 TBE running buffer.
6. Stain the gel in a 1 GelRed or SYBR Safe DNA Gel stain bath
for 15 min.
7. Using a UV transilluminator, analyze the quality of the RNAs
on the gel.
 Quantity and Quality Control of Small RNAs 23

3.2.2 Capillary 1. Prepare the ladder provided in the kit: Spin down the tube and
Electrophoresis transfer 10 μl of it to a RNase-free tube. Heat for 2 min at
70  C. Cool down on ice and add 90 μl of RNase-free water.
Prepare 5 μl aliquots using the Safe-Lock PCR Tubes provided
in the kit and store them at 70  C. Before use, thaw one tube
and keep it on ice (Fig. 1a) (see Note 8).
2. Equilibrate all solutions of the kit at room temperature for at
least 30 min before starting the experiments (see Note 6).
3. Place 550 μl (Agilent RNA 6000 Nano or Pico kit) or 650 μl
(Agilent Small RNA kit) of the gel matrix (red cap vial) into a
dedicated spin filter. Spin at room temperature for 10 min at
1500  g (Agilent RNA 6000 Nano or Pico kit) or for 15 min
at 10,000  g (Agilent Small RNA kit) (Table 1) (Fig. 1b).
4. Prepare 65 μl (Agilent RNA 6000 Nano or Pico kit) or 40 μl
(Agilent Small RNA kit) aliquots of the gel and store them at
4  C for a maximum of 1 month.
5. To avoid contamination of your RNA samples by RNases, clean
the electrodes before and after each experiment by filling one of
the wells of the electrode cleaner with 350 μl of fresh RNase-
free water (see Note 9).
6. Place the electrode cleaner in the Agilent 2100 bioanalyzer and
wait for 5 min before removing it.
7. Wait for 30 min to allow evaporation of the water from the
electrodes (see Note 10).
8. Prepare the gel–dye mix by adding 1 μl (Agilent RNA 6000
Nano or Pico kit) or 2 μl (Agilent Small RNA kit) of RNA dye
concentrate (blue cap vial) to a gel aliquot. Mix by pipetting up
and down and centrifuge for 10 min at 13,000  g at room
temperature (Fig. 1c).
9. During centrifugation, prepare your samples as follows: Dilute
your RNA samples with RNase-free water to be within the
optimal range concentration of the assay (Table 1).
10. Heat the diluted RNA samples for 2 min at 70  C to prevent
formation of secondary structures and cool them down on ice
(Fig. 1d).
11. Add 1 μl of your diluted RNA samples to 11 (or 12) tubes of
1.5 ml containing 5 μl of RNA marker (green cap vial) (see
Notes 11 and 12) (Table 1). Mix by pipetting up and down
(Fig. 1d).
12. Mix 1 μl of the ladder with 5 μl of RNA marker (green cap vial)
in a 1.5 ml tube (see Note 13) (Table 1). Mix by pipetting up
and down (Fig. 1d).
24 Virginie Marchand et al.

a) e)

1-3 G

4-6 G

7-9 G

Heat for 2 min Cool down 10-11 CS

at 70°C on ice

RNA Pico Chip

10 μl Add 90 μl of H2O Aliquot by 5 μl

ladder (RNase-free) (store at -70°C)

Load 9 μl of Gel-dye mix

G
b) G

CS

Close chip priming station

Centrifuge for Press the plunger of the syringe
Load on
10 min at 13,000 g at RT until it is hold by the clip
Wait for 30s
Release the clip and
550 μl Aliquot by 65 μl wait for the plunger to go up
gel matrix (store at 4°C) Open the chip priming station

Load 9 μl of Gel-dye mix

c) G

CS

+ 65 μl gel
aliquot
Centrifuge for
Load 9 μl of Conditioning Solution
10 min at 13,000 g at RT
G

G
1 μl Gel-dye mix ready
dye concentrate G

CS

d) Load 6 μl of Diluted Ladder

G

G
5 μl ladder G
aliquot Mix by pipetting
CS
up and down
6 μl diluted ladder

1 μl ladder
Load 6 μl of Diluted RNA sample
G

CS
+ + +
12 empty
1.5 ml tubes

Inspect for air bubbles or

liquid spilling in the wells

Heat for 2 min Mix by pipetting Place the chip in the

x μl at 70°C 1 μl up and down 6 μl diluted sample Agilent 2100 Bioanalyzer
(0.5-5 ng/μl) and start the run

Fig. 1 Quick guide for RNA Pico Chip preparation and loading. (a) Ladder preparation. (b) Gel preparation. (c)
Gel–dye mix preparation. (d) Samples and ladder preparation. (e) RNA Pico Chip loading
 Quantity and Quality Control of Small RNAs 25

13. Prepare the chip priming station before loading the gel–dye
mix. Check that the base plate is adjusted in its default position
(C position) (Fig. 1e).
14. Open a new syringe, slide it into the hole of the lock adapter
and screw it to the priming station. Adjust the plunger to 1 ml
(Fig. 1e).
15. Adjust the syringe clip to the highest top (Agilent RNA 6000
Nano or Pico kit) or to the lowest top (Agilent Small RNA kit)
position (see Table 1).
16. Once you are ready, load 9 μl of the gel–dye mix in the well
marked with a “G” surrounded by a black circle (see Note 14)
(Fig. 1e).
17. Close properly the chip priming station (see Note 15) and press
the plunger of the syringe until it is hold by the clip (Fig. 1e).
18. Wait exactly for 30 s (Agilent RNA 6000 Nano or Pico kit) or
60 s (Agilent Small RNA kit) and then release the clip.
19. Wait for 5 s until the plunger stops its upward move and pull it
slowly back to the 1 ml position.
20. Open the chip priming station and load 9 μl of gel–dye mix in
the other wells marked with a “G.”
21. Load 9 μl of the conditioning solution (not for the Agilent
RNA 6000 Nano kit) in the well marked “CS” (Fig. 1e).
22. Load 6 μl of the diluted ladder in the well marked with a ladder
(Fig. 1e).
23. Load 6 μl of the diluted RNA samples in the wells marked 1–11
(for Agilent RNA 6000 Pico and Small RNA kit) or 1–12 (for
Agilent RNA 6000 Nano kit) (Fig. 1e).
24. Inspect the chip and make sure that no liquid is present on the
edges of the wells. If any, pipet it away (see Note 14).
25. Insert the chip in the Agilent 2100 Bioanalyzer.
26. Carefully close the lid. The electrodes of the cartridge will enter
into the wells of the chip (see Note 15).
27. The 2100 expert software screen shows that you have inserted
a chip by displaying a chip icon on the top left corner of the
screen.
28. Select the appropriate assay you want to perform (e.g., eukary-
otic total RNA Pico series II).
29. Press “Start” to begin the chip run (see Note 16).
30. After the run, immediately remove the chip and clean the
electrodes (see Note 10).
31. Analyze the results of the chip (Fig. 2).
26 Virginie Marchand et al.

a A
RN tal R
NA
total to
t n
ec uma
ins h
Total RNA extracted from human PBMC cells (RIN=8.7)
[FU]
200

100

0
(nt)
25 500 4000 [nt] 4000
Total RNA extracted from insect cells (RIN=NA)
[FU] 2000
200 1000
500
200
0
25
25 500 4000 [nt]
RIN NA 8.7
b
Total RNA extracted from human PBMC cells (RIN=9.3)
[FU]
18S rRNA 28S rRNA
re
NA xtu
200 lR mi NA
ota NA m iR
100
sRNAs nt tt R
ed
ma a s rifi
hu ye pu
0
25 500 4000 [nt]
yeast tRNA mixture (RIN=2.6)
[FU]

1000
(nt)
500
4000
0
25 500 4000 [nt] 2000
Synthetic miRNA (RIN=2.6)
1000
[FU]
500
200

25
0
RIN 9.3 2.6 2.6
25 500 4000 [nt]

c [FU] Total RNA extracted from human PBMC cells (RIN=9.3)

50 miRNA tRNA
re
NA xtu
lR mi NA
ota NA m iR
0 nt t tR ed
ma as rifi
hu ye pu
4 20 60 100 150 [nt]

[FU] yeast tRNA mixture (RIN=2.6)

(nt)
0
4 20 60 100 150 [nt] 150

[FU] Synthetic miRNA (RIN=2.6)

100
200 80
60
100
40
0 20

4 20 60 100 150 [nt] 4

miRNA tRNA

Fig. 2 Examples of RNA profiles obtained using the Agilent 2100 Bioanalyzer. (a) High-quality insect or human
total RNA obtained after Trizol extraction analyzed on an RNA Pico Chip. The human total RNA trace profile is
 Quantity and Quality Control of Small RNAs 27

4 Notes

1. Several possible contaminants were tested in the concentration

range of 25–500 ng/ml. The corresponding data are provided
in the manufacturer’s protocol. For instance, presence of up to
0.01% SDS, 1% ethanol, 0.001% Triton X-100, 5 mM NaCl, or
1 mM MgCl2 in the RNA sample preparation is tolerated and
does not affect the accuracy of the quantification.
2. In both cases, if RNA degradation is observed, it is difficult to
assess whether this is due to poor quality of the RNA prepara-
tion or because RNases are present on the gel electrophoresis
device and degraded the sample while running. Incorporating
chlorine bleach (6% sodium hypochlorite) into a standard TAE
or TBE agarose gel helps preventing the latter problem [2].
3. The NanoDrop One is equipped with the “Acclaro Sample
Intelligence” technology. This feature helps to identify the
type of contaminant present in a sample, determine the level
of contamination, and obtain a corrected nucleic acid
concentration.
4. Make sure the sample is well centered on the bottom pedestal
and avoid air bubbles. Loading of 1.2 μl of RNA could be an
alternative to get more accurate and reproducible results.
5. Using RNase-free water instead of 10 mM Tris-EDTA (TE),
pH 8.0 to dilute your sample may lower the 260/280 ratio
below 2.0 due to the lower pH of water [7]. A 260/280 ratio
of 1.8 for samples diluted in RNase-free water is considered
“pure” for RNA.
6. The concentrated assay reagent contains DMSO, which is a
potential mutagen, therefore wear hand and eye protection and

Fig. 2 (continued) typical of a high-quality RNA since the 28S and 18S rRNA peaks are present and dominant.
A RIN (RNA Integrity Number) of 8.7 was attributed to the RNA sample, which is indicative of good quality. Note
the presence of abundant small RNAs including 5.8S and 5S rRNAs, tRNAs, and other noncoding RNAs ranging
from 25 to 200 nts. The insect total RNA trace profile is also typical of a high-quality RNA sample, even though
it looked like degraded and no RIN could be attributed since the 28S rRNA peak is absent. Indeed, upon heat
denaturation, the insect 28S rRNA is cleaved leading to fragments migrating with the 18S rRNA. (b) RNA trace
profiles obtained on a Pico RNA chip for a yeast total tRNA preparation and a synthetic miRNAs sample (21 nt)
were compared to a human total RNA preparation. As expected, yeast tRNA mixture and synthetic miRNA both
migrate as the small RNAs of the human total RNA preparation. (c) RNA trace profiles obtained on a Small RNA
chip for a yeast total tRNA preparation and a synthetic miRNAs sample (21 nt) were compared to a human total
RNA preparation. The human total RNA preparation leads to three major peaks, one between 50 and 80 nts,
corresponding mainly to tRNAs, one around 90 nts corresponding to snoRNAs, and one around 140 nts
corresponding to the 5.8S rRNA. 5S rRNA corresponds to a small pic at 120 nts. Though miRNAs are detected
in this sample, they represent too small a portion of total RNAs and are therefore not visible on the RNA trace
profile
28 Virginie Marchand et al.

be careful when preparing and handling reagents and samples.

Store this reagent at room temperature, desiccated and pro-
tected from light. Store the dilution buffer at room tempera-
ture and the standards at 4  C. All kit components are stable for
6 months.
7. In the protein kit, there are 3 standards and samples should be
incubated for 15 min instead of 2 min with kits dedicated to
nucleic acids.
8. We recommend doing this immediately upon kit arrival to
reduce the number of freeze and thaw events.
9. RNase contamination problems of the electrodes are quite
frequent and will affect the RIN of your sample. Therefore, if
the Agilent 2100 Bioanalyzer is also used for DNA assays, it is
strongly recommended to use a dedicated electrode cartridge
for RNA assays. In addition, we recommend for each chip to
load an internal RNA control (total RNA preparation with a
known RIN > 9). If you encounter contamination problems,
clean the electrode cartridge with an RNA Zap solution for at
least 10 min, then rinse the electrodes with RNase-free water
and let them dry out for at least one night.
10. Water on the electrodes will lead to abnormal RNA profiles.
Make sure any traces of water are evaporated before running
the chip.
11. To avoid RNase contamination problems, we recommend
labeling the RNA marker vial currently in use, and to split it
into aliquots. If you have any doubt concerning a possible
RNase contamination, trash the current vial and use a new
aliquot.
12. Do not leave any empty well otherwise the chip will not run
properly. If you do not have enough samples to load, use
RNase-free water instead and proceed as usual.
13. The ladder is quite stable at 70  C and may be used beyond
4 months.
14. The gel and samples loading steps on the chip are crucial. Insert
the tip of the pipette at the very bottom of the well when
loading gel or samples. This will prevent formation of air
bubbles. Placing the pipette at the wall or the edge of the
well will lead to poor results.
15. The chip priming station is properly closed only when you hear
a clear “click” sound.
16. The Agilent 2100 Bioanalyzer is very sensitive to vibrations,
therefore install it on a dedicated bench and make sure that no
vibrations will occur during the run.
 Quantity and Quality Control of Small RNAs 29

References
1. Rave N, Crkvenjakov R, Boedtker H (1979) Granzow M, Ragg T (2006) The RIN: an RNA
Identification of procollagen mRNAs trans- integrity number for assigning integrity values to
ferred to diazobenzyloxymethyl paper from RNA measurements. BMC Mol Biol 7:3
formaldehyde agarose gels. Nucleic Acids Res 5. Daniell H, Chebolu S, Kumar S, Singleton M,
6:3559–3567 Falconer R (2005) Chloroplast-derived vaccine
2. Aranda PS, LaJoie DM, Jorcyk CL (2012) antigens and other therapeutic proteins. Vaccine
Bleach gel: a simple agarose gel for analyzing 23:1779–1783
RNA quality. Electrophoresis 33:366–369 6. Winnebeck EC, Millar CD, Warman GR (2010)
3. Becker C, Hammerle-Fickinger A, Riedmaier I, Why does insect RNA look degraded? J Insect
Pfaffl MW (2010) mRNA and microRNA qual- Sci 10:159
ity control for RT-qPCR analysis. Methods 7. Wilfinger WW, Mackey K, Chomczynski P
50:237–243 (1997) Effect of pH and ionic strength on the
4. Schroeder A, Mueller O, Stocker S, Salowsky R, spectrophotometric assessment of nucleic acid
Leiber M, Gassmann M, Lightfoot S, Menzel W, purity. BioTechniques 22(474–476):478–481
 Chapter 4

Non-Coding RNA Silencing in Mammalian Cells by Antisense

LNA GapmeRs Transfection
Charbel Alfeghaly, Christelle Aigueperse, Sylvain Maenner,
and Isabelle Behm-Ansmant

Abstract
The assessment of non-coding RNAs (ncRNAs) functions highly relies on loss of function studies.
However, due to their exclusive or partial nuclear localization, many small and long ncRNAs are not
efficiently silenced by RNA interference. Antisense LNA GapmeRs constitute a good alternative to
RNAi. They allow an effective knockdown of ncRNAs with sizes greater than 80 nucleotides, regardless
of their cellular localization. This chapter focuses on the silencing of two different nuclear ncRNAs (ANRIL
and SATIII RNAs) in mammalian cells using antisense LNA GapmeRs with two different transfection
methods: calcium phosphate-mediated transfection and LipofectamineTM 2000.

Key words lncRNA silencing, Antisense LNA GapmeRs, Calcium phosphate transfection, Lipofecta-
mineTM 2000, ANRIL, SATIII

1 Introduction

Despite recent advances in our understanding of noncoding RNAs’

roles, most of them and more especially long noncoding RNAs
(lncRNAs) functions remain poorly understood. Loss-of-function
analyses are essential in deciphering the functions of ncRNAs by
assessing the biological consequences of suppressing their expres-
sion. However, many ncRNAs are confined to the nucleus and thus
are inefficiently targeted by the cytoplasmic RNA-induced silencing
complex (RISC) [1, 2]. To overcome this limitation, antisense LNA
GapmeRs were designed to provide an effective knockdown of both
coding and noncoding RNAs with a size greater than 80 nucleo-
tides, regardless of their cellular localization. Antisense LNA Gap-
meRs are single-stranded antisense oligonucleotides (ASOs) that
catalyze nuclear RNase H-dependent degradation of complemen-
tary RNA targets (Fig. 1). They are 16 nucleotides long and are
enriched with LNA (Locked Nucleic Acids) nucleotides in the
flanking regions and DNA nucleotides in an LNA-free central

Mathieu Rederstorff (ed.), Small Non-Coding RNAs: Methods and Protocols, Methods in Molecular Biology, vol. 2300,
https://doi.org/10.1007/978-1-0716-1386-3_4, © Springer Science+Business Media, LLC, part of Springer Nature 2021

31
32 Charbel Alfeghaly et al.

Transcription

ncRNA
LNA DNA LNA
Antisense LNA GapmeR
Nucleus
RNaseH
RNase
RNase H cleavage

Degraded ncRNA

Fig. 1 Silencing of nuclear noncoding RNA using antisense LNA GapmeRs.

Antisense LNA GapmeRs are single-stranded oligonucleotides containing a
DNA portion flanked by LNA. They hybridize by complementarity to their RNA
targets. The DNA:RNA duplexes are recognized by nuclear RNase H, creating an
endonucleolytic cleavage in the RNA strand. The two generated fragments are
degraded by exonucleases. The antisense LNA GapmeR is then released and will
guide degradation of another RNA strand. (Adapted from (https://www.qiagen.
com))

gap. LNA are a class of high-affinity RNA analogs in which the

ribose ring is “locked” in the ideal conformation for Watson–Crick
binding thanks to a 2-O,40 -C-methylene bridge [3]. As a result,
LNA-containing oligonucleotides exhibit higher thermal stability
(Tm) when hybridized to a complementary DNA or RNA strand,
regardless of the GC contents. Moreover, incorporation of LNA
bases suppresses innate immune stimulation [4] and confers resis-
tance to endonucleases and exonucleases, which leads to higher
in vitro and in vivo stability. This resistance to enzymatic degrada-
tion is further increased by the fact that antisense LNA GapmeRs
have fully modified phosphorothioate (PS) backbones. Moreover,
antisense LNA GapmeRs work independently of the RISC complex
and thus have no miRNA-like off-target effects. As they are single
stranded, they guarantee strand-specific knockdown, which is of
particular interest when studying ncRNAs that derive from tran-
scriptionally complex loci with overlapping sense and antisense
transcripts.
 Non-Coding RNA Silencing 33

Due to their small size and stability in culture media, antisense

LNA GapmeRs can be taken up by cells without the need for a
transfection agent. Nevertheless, non-assisted uptake does require
higher concentrations of antisense LNA GapmeR than would be
needed when using transfection agents, and the knockdown kinet-
ics would be slower. In this chapter, we describe the two transfec-
tion protocols commonly used in our lab to efficiently transfect
antisense LNA GapmeRs. First, calcium phosphate-mediated trans-
fection, a biochemical method, is a simple handling and inexpensive
procedure. Calcium phosphate forms an insoluble coprecipitate
with DNA, which is absorbed by endocytosis or phagocytosis by
the cells [5]. This technique has proven to be very effective for the
delivery of oligonucleotides (siRNA, ASOs) in our hands. Another
delivery system is LipofectamineTM 2000, a liposomal cationic lipid
reagent, which forms a complex with the DNA. The resulting stable
cationic complexes adhere to the negatively charged cell membrane
before fusing with them [6]. LipofectamineTM 2000 is known to be
a highly efficient carrier of LNA oligonucleotides to cells nuclei [7].
In this chapter, we illustrate these efficient transfection meth-
ods by silencing two nuclear lncRNAs: SATIII and ANRIL. SATIII
lncRNAs are transcribed from the pericentromeric SATIII repeats
located on human chromosomes 9, 12, 15 [8]. Rosic et al. [9]
showed that Drosophila melanogaster SATIII RNAs, on the X chro-
mosome, constitute an integral part of centromere identity and are
essential for kinetochore formation and cell division. In addition,
SATIII lncRNAs promote intron retention during thermal stress
recovery [10]. On the other hand, the ANRIL lncRNA is tran-
scribed from the 9p21 locus and is alternatively spliced into several
isoforms harboring different combination of exons. ANRIL was
shown to promote in cis the epigenetic silencing of the CDKN2A
and CDKN2B tumor suppressor genes and hence to promote cell
proliferation [11, 12]. Using calcium phosphate-mediated trans-
fection, we efficiently silenced the expression of human SATIII and
ANRIL lncRNAs (Fig. 2a, b). Rapid effects were observed for
SATIII silencing as extinction appeared as soon as 6 h after treat-
ment with antisense LNA GapmeRs. On the contrary, 48 h were
needed to reach an efficient ANRIL silencing using two consecutive
treatments with antisense LNA GapmeRs independently of the
transfection method used (Fig. 2b, c).
To conclude, antisense LNA GapmeRs containing a central
DNA segment flanked by LNA gaps offer an alternative to
siRNA-dependent technology for suppression of nuclear noncod-
ing RNA expression, with the calcium phosphate-mediated trans-
fection being the most efficient and cheapest transfection method.
 34 Charbel Alfeghaly et al.

A. B. C.
100 LNA CTL 100 LNA CTL 100 LNA CTL
LNA SATIII 6h LNA ANRIL LNA ANRIL

Relative RNA expression (%)

Relative RNA expression (%)

Relative RNA expression (%)

LNA SATIII 24h n=3 n=1

80 80 80
n=3

60 60 60

40 40 40
19 25
19.9
20 20 20
4.8
0 0 0
SATIII ANRIL ANRIL

Calcium phosphate Lipofectamine

Fig. 2 Silencing of SATIII and ANRIL lncRNAs using specific antisense LNA GapmeRs. (a) Calcium phosphate-
mediated transfection of HeLa S3 cells is used to deliver either LNA GapmeRs specifically targeting SATIII RNA
(LNA SATIII) or LNA oligonucleotides against the GFP gene (LNA CTL) as a negative control. The efficiency of
SATIII knockdown is monitored by RT-qPCR. Transfection of SATIII LNA dramatically reduces the expression
level of SATIII RNAs and this effect is greater 6 h post-transfection compared to 24 h. Values are calculated
from three independent experiments and normalized against GAPDH mRNA level. The level of the SATIII RNA is
expressed relatively to the level of the control (100%). (b) Calcium phosphate-mediated transfection of
HEK293 cells is used to deliver either LNA GapmeRs specifically targeting ANRIL (LNA ANRIL) or a scrambled
sequence (LNA CTL) as a negative control. Efficiency of ANRIL knockdown is monitored by RT-qPCR.
Transfection of LNA ANRIL reduces ANRIL expression up to 80% 48 h post-transfection. Values are calculated
from three independent experiments and normalized against GAPDH mRNA levels. The level of ANRIL is
expressed relatively to the level of the control (100%). (c) Lipofectamine 2000 is used to transfect HEK293
cells with either LNA GapmeRs specifically targeting ANRIL (LNA ANRIL) or a scrambled sequence (LNA CTL) as
a negative control. Efficiency of ANRIL knockdown is monitored by RT-qPCR, which shows an efficient
reduction in ANRIL’s expression up to 75%, similarly to the calcium phosphate method. Values are calculated
from one experiment and normalized against GAPDH mRNA levels. The level of ANRIL is expressed relatively to
the level of the control (100%). Means  SEM are shown

2 Materials

2.1 Mammalian Cell 1. HeLa S3 cells and HEK293 cells (ATCC) (see Note 1).
Culture 2. DMEM (Dulbecco’s Modified Eagle Medium) cell culture
medium, supplemented with 10% of fetal calf serum, 2 mM
L-glutamine, 100 U/ml of penicillin, and 100 μg/ml of
streptomycin.
3. Six-well plates (for 100-mm dishes, reagent quantities are mul-
tiplied by 6).
 Non-Coding RNA Silencing 35

2.2 Antisense LNA Chemically modified antisense LNA GapmeRs are produced by
GapmeRs Exiqon-Qiagen, resuspended in nuclease-free H2O to a concentra-
(Exiqon-Qiagen) tion between 50 and 100 μM (see Note 2) and stored as aliquots at
20  C to avoid repeated freeze and thaw cycles.

2.3 Standard 1. TE (0.1, pH 7.6): 1 mM Tris–HCl, pH 8.0, 0.1 mM EDTA.

Calcium Phosphate 2. 2 M calcium chloride (CaCl2). Sterilize by filtration and store
Procedure frozen as 1 ml aliquots at 20  C.
3. 2 HEPES-buffered saline (HBS): 50 mM HEPES, 0.28 M
NaCl, 10 mM KCl, 1.5 mM Na2HPO4, pH 7.05 (see Note 3).
Sterilize by filtration and store frozen as 50 ml aliquots at
20  C.

2.4 Lipofectamine 1. Lipofectamine™ 2000 Transfection Reagent (Life

2000 Procedure Technologies).
2. Gibco™ Opti-MEM™ I Reduced Serum Medium (Life
Technologies).

3 Methods

3.1 Calcium 1. One day prior to transfection, seed 3  105 exponentially

Phosphate growing cells in 2 ml DMEM medium in 6-well plates. Incu-
Transfection bate cells for 20–24 h at 37  C with 5% CO2 (see Note 4).
2. One to 5 h before transfection, replace the medium with 2 ml
of fresh growth medium.
3. For each individual transfection in 6-well plates, dilute 3 μl of
50 μM SATIII antisense LNA GapmeRs or 1 μL of 50 μM
ANRIL antisense LNA GapmeRs with 0.1X TE to a final
volume of 36.8 μl and combine with 5.2 μl of 2 M CaCl2.
4. Add an equal volume of 2 HBS (42 μl in this case) with a
pipette.
5. Mix by pipetting up and down about 10 times to inject air
bubbles, which enhances the formation of the transfection
precipitate.
6. Incubate for 20 min at room temperature (see Note 5).
7. Add the 84 μl of transfection mixture drop by drop in each well
(see Note 6).
8. After 4–16 h of incubation, remove the DNA precipitate con-
taining medium and replace it with warm complete growth
medium (see Note 7).
9. Return the cells to the incubator for 2–96 h at 37  C with 5%
CO2 (see Note 8 and 9).
36 Charbel Alfeghaly et al.

3.2 Lipofectamine™ 1. One day prior to transfection, seed 5  105 exponentially

2000 Transfection growing cells in 2 ml of DMEM medium in 6-well plates.
Incubate cells for 20–24 h at 37  C with 5% CO2 (see Note 10).
2. One to 5 h before transfection, replace the medium with 2 ml
of fresh growth medium.
3. For each individual transfection in 6-well plates, dilute 1 μl of
50 μM antisense LNA GapmeRs with 249 μL of Gibco™ Opti-
MEM™ I Reduced Serum Medium. Mix well by pipetting.
4. In a separate tube, dilute 5 μL of Lipofectamine™ 2000
reagent in 245 μL of Gibco™ Opti-MEM™ I Reduced
Serum Medium. Mix well by pipetting.
5. Incubate for 5 min at room temperature.
6. Add the Lipofectamine mix to the solution containing the LNA
GapmeRs and mix gently by pipetting.
7. Incubate for 20 min at room temperature.
8. Add the 500 μl of transfection mixture drop by drop in
each well.
9. Return the cells to the incubator for 24–48 h at 37  C with 5%
CO2 (see Note 8).

4 Notes

1. Other cell lines can easily be transfected as well.

2. A master mix can be assembled if multiple transfections of the
same antisense LNA GapmeRs have to be performed. For
ANRIL, a mix of 4 LNA GapmeRs hybridizing to different
ANRIL regions was used at 12.5 μM each (50 μM total).
3. Reagent consistency is critical to achieve a high transfection
efficiency and reproducibility; small changes in pH can affect
transfection efficiency.
4. It is critical that the cell density ranges between 30% and 60%
confluency at the day of transfection.
5. Longer incubation times can result in the formation of fewer
but larger precipitates that reduce transfection efficiency.
6. For the depletion of SATIII and ANRIL lncRNAs, cells were
transfected with 75 nM and 25 nM of antisense LNA Gap-
meRs, respectively, into 2 ml of medium. The concentration of
antisense LNA GapmeRs to be used is usually in the 1–100 nM
range and depends on the abundance of the targeted RNA in
the cells.
7. Prolonged cell contact with calcium phosphate precipitates
may result in cytotoxicity.
 Non-Coding RNA Silencing 37

8. Antisense LNA GapmeRs should be tested to find out the best

experimental conditions to get a maximal silencing efficiency. If
the effect is too weak, you can proceed with a second transfec-
tion after 24–48 h to prolong the effect. If necessary, cells
should be split prior to the second transfection. For the deple-
tion of ANRIL, two rounds of transfection were done indepen-
dently of the transfection method used. The second round was
performed 24 h after the first one. Cells were collected 48 h
after the second transfection.
9. For the depletion of SATIII RNAs, 6 h of transfection were
sufficient.
10. It is critical that the cell density ranges between 50% and 70%
confluency on the day of transfection.

Acknowledgments

This work was supported partly by the french PIA project “Lorraine
Université d’Excellence” (ANR-15IDEX-04-LUE) and the RHU
program FIGHT-HF (ANR-15-RHU-004). The CNRS and UL
are also thanked for fundings.

References
1. Liang XH, Vickers TA, Guo S, Crooke ST analog in therapeutics and biotechnology. Oli-
(2011) Efficient and specific knockdown of gonucleotides 14:130–146
small non-coding RNAs in mammalian cells 8. Biamonti G, Vourc’h C (2010) Nuclear stress
and in mice. Nucleic Acids Res 39(3):e13 bodies. Cold Spring Harbor Perspectives in
2. Ploner A, Ploner C, Lukasser M, Biology
Niederegger H, Huttenhofer A (2009) Meth- 9. Rosic S, Köhler F, Erhardt S (2014) Repetitive
odological obstacles in knocking down small centromeric satellite RNA is essential for kinet-
noncoding RNAs. RNA 15:1797–1804 ochore formation and cell division. J Cell Biol
3. Singh SK, Koshkin AA, Wengel J, Nielsen P 207:335–349
(1998) LNA (locked nucleic acids): synthesis 10. Ninomiya K, Adachi S, Natsume T, Iwakiri J,
and high-affinity nucleic acid recognition. Terai G, Asai K, Hirose T (2019) LncRNA-
Chem Commun 1998:455–456 dependent nuclear stress bodies promote
4. Judge AD, Bola G, Lee AC, MacLachlan I intron retention through SR protein phosphor-
(2006) Design of noninflammatory synthetic ylation. EMBO J 29:e102729. https://doi.
siRNA mediating potent gene silencing org/10.15252/embj.2019102729
in vivo. Mol Ther 13:494–505 11. Kotake Y, Nakagawa T, Kitagawa K, Suzuki S,
5. Graham FL, van der Eb AJ (1973) A new tech- Liu N, Kitagawa M, Xiong Y (2011) Long
nique for the assay of infectivity of human ade- non-coding RNA ANRIL is required for the
novirus 5 DNA. Virology 52:456–467 PRC2 recruitment to and silencing of
6. Felgner PL, Gadek TR, Holm M, Roman R, p15INK4B tumor suppressor gene. Oncogene
Chan HW, Wenz M, Northrop JP, Ringold 30:1956–1962
GM, Danielsen M (1987) Lipofection: a highly 12. Yap KL, Li S, Muñoz-Cabello AM, Raguz S,
efficient, lipid-mediated DNA-transfection Zeng L, Mujtaba S, Gil J, Walsh MJ, Zhou
procedure. Proc Natl Acad Sci 84:7413–7417 M-M (2010) Molecular interplay of the non-
7. Jepsen JS, Sorensen MD, Wengel J (2004) coding RNA ANRIL and methylated histone
Locked nucleic acid: a potent nucleic acid H3 lysine 27 by Polycomb CBX7 in transcrip-
tional silencing of INK4a. Mol Cell
38:662–674
 Part II

Detection of Small Non-Coding RNAs

 Chapter 5

Northern Blot Detection of Tiny RNAs

Jana C. Wiegard, M. Amri C. Schlüter, Olga Y. Burenina,
Elena A. Kubareva, Gabriele Klug, Arnold Grünweller,
and Roland K. Hartmann

Abstract
Successful detection of very small RNAs (tiny RNAs, ~8–15 nt in length) by northern blotting depends on
tailored protocols with respect to transfer and immobilization on membranes as well as design of sensitive
detection probes. For RNA crosslinking to positively charged membranes, we compared UV light with
chemical RNA crosslinking by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), using either
denaturing or native polyacrylamide gels. We show that northern blot detection of tiny RNAs with
50 -digoxigenin-labeled DNA/LNA mixmer probes is a highly sensitive and specific method and, in our
hands, more sensitive than using a corresponding DNA/LNA mixmer probe with a 50 -32P-end-label.
Furthermore, we provide a robust protocol for northern blot analysis of noncoding RNAs of intermediate
size (~50–400 nt).

Key words Northern blot, EDC crosslinking, UV crosslinking, Digoxigenin, LNA, Native PAGE

1 Introduction

Recent advances in high-throughput sequencing methods and bio-

informatics have led to the identification of new classes of noncod-
ing RNAs (ncRNAs) with regulatory function in bacteria, archaea
and eukaryotes. However, expression patterns and proof of func-
tionality of such ncRNAs need to be confirmed with established
standard techniques including RT-qPCR, microarrays, genetic
methods, and, of course, northern blotting. The detection of very
small RNAs (miRNAs and shorter ones, termed “tiny RNAs” in the
following) poses a challenge, as it requires highly sensitive and
specific methods to confirm their identity. This prompted the
development of specialized RT-qPCR techniques, such as the
stem-loop primer method, to quantify individual miRNAs

Jana C. Wiegard and M. Amri C. Schlüter are joint first authors.

Mathieu Rederstorff (ed.), Small Non-Coding RNAs: Methods and Protocols, Methods in Molecular Biology, vol. 2300,
https://doi.org/10.1007/978-1-0716-1386-3_5, © Springer Science+Business Media, LLC, part of Springer Nature 2021

41
42 Jana C. Wiegard et al.

[1]. Nevertheless, northern blotting remains indispensable for a

direct visualization of cellular RNAs and their length variants rang-
ing from primary transcripts over processing intermediates to
mature forms. A problem for the detection of tiny RNAs by north-
ern blotting is their limited interaction surface, thus increasing the
risk that their covalent immobilization on hybridization mem-
branes may sterically hinder accessibility of the probe. Another
risk is that tiny RNAs may pass through the membrane instead of
being retained on it during the blotting procedure. Improved pro-
tocols for the detection of small RNAs have already been described
to overcome several limitations of conventional northern blot pro-
cedures [2, 3], which were further refined for the detection of tiny
RNAs [4, 5]. We previously described an approach combining
native polyacrylamide gels, RNA immobilization on nylon mem-
branes by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
(EDC) crosslinking, and non-radioactive 50 -digoxigenin (DIG)-
end-labeled oligonucleotide probes containing locked nucleic acid
(LNA) modifications for the detection of tiny RNAs with high
sensitivity and specificity [4–7].
In this protocol, we first describe some preparatory steps for
tiny RNAs used as size markers for northern blot experiments, such
as their 50 -phosphorylation and quality control by denaturing
PAGE and different staining methods. For ncRNAs of intermediate
size (~50–400 nt), a procedure involving denaturing PAGE, UV
crosslinking, and DIG-labeled T7 transcript probes is described.
For tiny RNAs (~8–15 nt), we provide protocols for 20% denatur-
ing or native PAGE, RNA immobilization on the membrane by UV
or EDC crosslinking, and detection by 50 -DIG-labeled or 5-
0 32
- P-labeled DNA/LNA-mixmer probes.

2 Materials

Prepare all solutions with autoclaved ddH2O and analytical grade

reagents for use in molecular biology. Prepare reagents at room
temperature, store them at room temperature (short-term storage)
or in aliquots at 20  C (long-term storage) if not stated
otherwise.

2.1 50 -Phos- 1. T4 polynucleotide kinase (PNK) (10 U/μl).

phorylation of RNA 2. 10  T4 polynucleotide kinase buffer: 500 mM Tris–HCl
Oligonucleotides (pH 7.6 at 25  C), 100 mM MgCl2, 50 mM DTT, 1 mM
spermidine.
3. 10 mM ATP in ddH2O.
 Northern Blotting of Tiny RNAs 43

2.2 RNA GelRed structurally consists of two ethidium subunits that are
Oligonucleotide bridged by a linear oxygenated spacer and penetrates the skin less
Staining with GelRed effectively than ethidium bromide (EtBr), thus being less toxic [8].
1. Stock solution: 10,000  concentrated GelRed Acid Stain.
2. Working solution: Supplement 100 ml of 1  TBE with 10 μl
of GelRed stock solution; solution can be used repeatedly until
staining efficiency drops.

2.3 RNA 1. Methylene blue (crystalline powder).

Oligonucleotide 2. Working solution: Prepare a 0.04% (w/v) solution in ddH2O;
Staining solution can be used repeatedly until staining efficiency drops.
with Methylene Blue

2.4 PAGE 1. 5  TBE electrophoresis buffer: 445 mM Tris base, 445 mM

and Transfer of RNA boric acid, 10 mM EDTA.
to Nylon Membranes 2. Native polyacrylamide gel: 1  TBE, polyacrylamide/bisacry-
lamide (24:1); for polymerization, add 1/100 volume 10%
(w/v) ammonium persulfate (APS) and 1/1000 volume of
N,N,N0 ,N0 -tetramethylethylenediamine (TEMED) to the
acrylamide gel solution.
3. Denaturing polyacrylamide gel: 1  TBE, polyacrylamide/
bisacrylamide (24:1), 8 M urea. For polymerization, add 10%
APS and TEMED as in point 2 before.
4. 2  native loading buffer: 0.025% (w/v) bromophenol blue,
0.025% (w/v) xylene cyanol blue, 20% (v/v) glycerol.
5. 2  denaturing loading buffer: 0.02% (w/v) bromophenol
blue, 0.02% (w/v) xylene cyanol blue, 8 M urea, 50% (v/v)
formamide, 2  TBE; alternatively: 0.025% (w/v) bromophe-
nol blue, 0.025% (w/v) xylene cyanol blue, 95% (v/v) form-
amide, 0.025% (w/v) SDS, 0.5 mM Na2EDTA adjusted to
pH 8.0 with NaOH.
6. Whatman paper, 1.5 mm thickness.
7. Positively charged nylon membrane, 10  15 cm: microporous
nylon 66 membrane on a polyester support, carrying positively
charged quaternary ammonium groups; surface properties:
0.45 μm pore size. We use the nylon membranes from
Merck/Sigma-Aldrich (No. 11209299001).
8. Semidry blotter (see Note 1).

2.5 UV and EDC 1. UV crosslinker. We use a Stratagene UV Stratalinker 1800 with

Crosslinking 254 nm bulbs.
for Fixation of RNAs 2. 12.5 M 1-methylimidazole stock solution.
on Membranes
44 Jana C. Wiegard et al.

3. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochlo-

ride, also termed N-(3-dimethylaminopropyl)-N0 -ethylcarbo‐
diimide hydrochloride (EDC).
4. Saran film.

2.6 Probe Generation 1. 10  DIG labeling mix (Merck).

by T7 Transcription 2. T7 RNA polymerase.
3. 10  transcription buffer: 400 mM Tris–HCl (pH 8.0 at
20  C), 60 mM MgCl2, 100 mM DTT, 20 mM spermidine.
4. Bacillus subtilis antisense 5S rRNA (~115 nt). For the tran-
scription of antisense 5S rRNA, a PCR template of ~130 bp
is used.
5. B. subtilis antisense 6S-1 RNA (~190 nt). For the transcription
of antisense 6S-1 RNA, a linearized pUC18 plasmid derivative
is used.
6. 200 mM EDTA solution, pH 8.0. Sterilize by filtration and
store at 20  C.

2.7 Hybridization 1. RNA p146S-1: 50 -GUU CGG UCA AAA CU-30 .

and Detection 2. RNA p86S-1: 50 -GUU CGG UC-30 .
Procedures
3. RNA p146S-2: 50 -AAA GGU UAA AAC UU-30 .
4. RNA p156S-2: 50 -AAA GGU UAA AAC UUA-30 (synthesized
at Lomonosov Moscow State University [9]).
5. RNA p206S-2: 50 -AAA GGU UAA AAC UUA AUU CA-30
(synthesized at Lomonosov Moscow State University [9]).
These RNAs are HPLC-purified and desalted, with
50 -OH ends.
6. Detection probes:
– probe p146S-1: 50 -DIG-aGt tTt gAc cGa Ac-30 .
– probe p86S-1: 50 -DIG-GAC cGA AC-DIG-30 .
– probe DIG6S-2: 50 -DIG-gTt tTa aCc tTt-30 .
– probe 32P6S-2: 50 -taa gTt tTa aCc tTt-30 .
DNA residues are indicated by lower and LNA residues by
upper case letters; DIG ¼ digoxigenin. These probes are
HPLC-purified and desalted.
7. Hybridization solution: DIG Easy Hyb Granules (Merck) (see
Note 2) [5].
8. Hybridization oven.
9. 20  SSC: 3 M NaCl, 0.3 M sodium citrate, pH 7.0.
10. Stringent wash buffer I: 2  SSC, 0.1% (w/v) SDS.
11. Stringent wash buffer II: 0.1  SSC, 0.1% (w/v) SDS.
 Northern Blotting of Tiny RNAs 45

12. 10  maleic acid buffer: 1 M maleic acid, 1.5 M NaCl, pH 7.5.

13. 10  blocking solution (Merck). Diluted 1:10 in 1  maleic
acid buffer.
14. Antibody solution: 1  blocking solution with Anti-
Digoxigenin-AP Fab fragments (coupled to alkaline phospha-
tase) diluted 1:10,000 (Merck).
15. 10  wash buffer (Merck).
16. Detection buffer: 0.1 M Tris–HCl, 0.1 M NaCl, pH 9.5.
17. Disodium 2-chloro-5-(4-methoxyspiro {1,2-dioxetane-3,2-
0
-(50 -chloro)tricyclo[3.3.1.13,7]decan}-4-yl) phenyl phos-
phate (CDP-Star, chemiluminescent substrate for alkaline
phosphatase (Sigma-Aldrich)).
18. Kodak BioMax Light Film and commercially available devel-
oper and fixer solutions.
19. Alternatively, BioRad ChemiDoc Imaging System according to
the manufacturer’s instructions.
20. 250  0.05 mm transparent plastic (polyethylene) tube film.
21. Plastic bag sealer.
22. Phosphorimager: Fuji FLA-3000 R and the software AIDA,
version 3.45 (Fujifilm) or equivalent.
23. Stripping buffer: 50% formamide, 50 mM Tris–HCl pH 7.5,
5% (w/v) SDS (prepare freshly).

3 Methods

3.1 50 -Phos- 1. Add 5 μl of 10  T4 polynucleotide kinase (PNK) buffer and

phorylation 5 μl of 10 mM ATP to 300 pmol of 50 -OH RNA oligonucleo-
of Chemically tide and adjust the volume to 47.5 μl with ddH2O.
Synthesized Marker 2. Add 2.5 μl of T4 PNK (10 U/μl).
RNA Oligonucleotides 3. Incubate for 1 h at 37  C.
4. Add 5 μl of ATP and 2.5 μl of T4 PNK. Incubate for another
1 h at 37  C. Transfer the sample to 20  C for storage.
5. Aliquots of the sample can be directly loaded onto PAA gels for
northern blot hybridization (see Note 3).

3.2 Staining Short RNA oligonucleotides may not be stained by intercalating

of Tiny RNAs dyes (EtBr or GelRed), particularly if shorter than ~10 nt (see
example in Fig. 1a). In such cases, staining by Methylene blue is
the method of choice.
1. After electrophoresis, submerge the gel in GelRed or Methy-
lene blue working solution for 15–30 min (GelRed) or 45 min
(Methylene blue).
Another Random Scribd Document
with Unrelated Content
allowing these superior methods to be observed and studied by the
less able.
A conscious sense of inferiority is also no obstacle, for a mother
having that feeling would be eager to improve by study of the better
ways.
These six mothers divide the working days of the week among them,
agreeing that each shall on her chosen day take charge of the
children of the other five. This might be for a part of the day or the
whole day, as is thought best,—let us suppose it merely for the
afternoon; and it could be limited, as desired, to children of a certain
age, and still further reduced, as a mild beginning, to one child
apiece from each family.
This would give, as a minimum, five extra children on one afternoon
a week to each mother. The maximum would be of course uncertain;
but, if all the children of each mother were thus to go visiting for any
part of the day, it would give to each one day in which that larger
responsibility was undertaken, and five days free. There would
remain Sunday, in which each family, complete, would be at home.
Now let us take a hypothetical case, and suppose that our six
mothers, with considerable trepidation, have chosen one child apiece
that they were willing to intrust for the afternoon to the watchful
care of these familiar friends. The children, be it rigidly insisted, are
to know nothing whatever of the purposes or methods involved. All
that little Johnny Black knows is that Mrs. White has asked him to
come over on Monday afternoon and play with Alice and Billy White,
and some other children that he knows, too; that presently Mrs.
Green has them come to her house on Tuesday, and Mrs. Brown on
Wednesday; that his mamma lets them all come and play with him
on Thursday,—in short, that his afternoons have become full and
rich and pleasantly exciting, like some wonderful procession of
parties.
"Not like regular parties, either," Johnny would explain. "You don't
have to dress up—much,—just be clean, to begin with. And they
don't have ice-cream and macaroons,—only just milk and crackers
when you get hungry; and—well, 'tisn't so much regular games and
p'r'aps dancin'—like a party,—we just play. And Mrs. White, or
whichever one 'tis, she generally has some nice young lady in with
her; and they sort of keep things going,—as if 'twas a real party. It's
nicer some ways, I think."
"And which place do you like best, Johnny?"
"Oh, I do' know! Billy White has the biggest yard. But Jim Grey has
the best swing; and there's a pond at Susy Green's,—a real pond,—
and nothing but girls live there! Then it's lots of fun when they come
to our house, 'cause I can show 'em my rabbits and make Jack do all
his tricks."
Yes, the children all enjoy it. It means variety, it means company, it
means a wider and closer acquaintance and all the benefits of well-
chosen association and larger environment. It fills a part of the day.
There is no more aimless asking, "What shall I do now?" with the
vague response, "Oh, run away and play!" or the suggestion of some
well-worn amusement.
It means, too, a little more sense of "company manners" and
behaviour, and, on the other hand, a better appreciation of home
life.
And to the mother,—what good will this do her?
Each mother would have one day in the week in which to carefully
observe children,—not her own specially beloved children, but just
children, as such. Her observation and care should be absolutely
unobtrusive: the moment the little ones knew they were being
watched, the value of the plan would be greatly impaired; and, to
stop at a minor detail, from the palpable necessity for doing this
work without the child's consciousness, mothers would learn to
cover the machinery of government at home. It is one of our
grossest and most frequent errors in the management of children
that we openly discuss our efforts and failures. They know that we
are struggling to produce certain results in their behaviour, usually in
a futile manner.
With, however, a large and definite purpose resting so absolutely on
the child's unconsciousness, more wisdom in this line would soon
develope.
The mother who now says, "What would you do with a child like
that?" or "I'm sure I don't know what to do with that child!" before
the child in question, would soon perceive that such an attitude in an
educator does not produce confidence in the object of the
education. Quietly and unostentatiously, and often with the
assistance of some keen girl-friend, these mothers would soon learn
to observe accurately, to generalise carefully, to reduce cautiously,
and then to put the deduction into practice and observe the results.
As beginners, pioneers, they should make their first steps very
modestly. For the first season some one trait should be chosen for
study,—say self-control or courage or consideration of others. Having
decided on their line of observation, let each mother make a little
note of how high each child in the group stands in this line.
How much self-control has my Johnny, as measured by his age?—as
compared with others of his age? When did I first notice self-control
in Johnny? When have I seen it greatest? Does he gain in it? What
should be done to help Johnny gain in self-control? And then go over
the same questions with regard to the other children.
Then, with self-control as the characteristic, the natural development
and best education of which they wish to study, the afternoon
parties begin. At first the children might be left absolutely free to
play in ordinary lines. Then, after the first observations were
recorded, delicate experiments could be introduced, and their results
added to the record.
It is very difficult for the individual mother to rightly estimate her
own children. "Every crow thinks her babe the blackest."
Yet the character of the child is forming without regard to any fond
prejudice or too severe criticism; and his life's happiness depends on
his interaction with people in general, not simply with beloved ones
at home. The measure of Johnny's self-control may not seem
important to the parental love which covers or the parental force
which compels; but to Johnny's after-life its importance is pre-
eminent. When one sits for a portrait to a fond and familiar friend,
and sees all fondness and familiarity die out from the eyes of the
artist, feels one's personality sink into a mass of "values," it brings a
strange sense of chill remoteness. So, no doubt, to the mother heart
the idea of calmly estimating Johnny's self-control and comparing it
with Jim Grey's seems cold enough. To have Mrs. Grey estimate it,—
and perhaps (terrible thought!) to estimate it as less than Jim's,—
this is hard, indeed.
Yet this is precisely what is to be obtained in such a combination as
this, and in no other way,—the value of an outside observer, through
Mrs. Grey's estimate.
Nobody's opinion alters facts. The relative virtues of Johnny and Jim
remain unchanged, no matter what their respective mothers think or
what their irrespective mothers think. But each mother will derive
invaluable side-lights from the other mother's point of view.
Each opinion must be backed with illustration. Instances of observed
behaviour must be massed before any judgment has value.
"I think your Jim is so brave, Mrs. Grey. When the children were with
me the other day, the cow got loose; and the girls all ran. Some
boys ran, too; and Jimmy drove her back into the cow-yard."
"But Jimmy was the oldest," says Mrs. White. "Perhaps, if he'd been
as young as my Billy, he wouldn't have been so brave."
"And he is afraid of the dark," says Mrs. Brown. "At my house he
wouldn't go into the back cellar after apples, even with the other
children. Isn't he afraid of the dark, Mrs. Grey?"
Mrs. Grey admits this, but cites instances to show courage in other
directions. And always five dispassionate observers to the one
deeply loving and prejudiced.
If it should happen that Jimmy is generally admitted brave beyond
his years, with the one exception of fearing darkness, and that
exception traceable to a nurse-maid's influence, the mother of
Jimmy is rejoiced; and a strong light is thrown on the nurse
question. If it prove that by general opinion there is a lack of
courage such as should belong to his years, there is cause for special
study and special action in this line. Most valuable of all, the habit of
observing a child's behaviour as an expression of character is
formed.
The six mothers would of course meet to compare notes, preferably
in evenings, when children were all in bed and fathers could be
present; and the usual difficulty of leaving home in the evening
could be met in such an important case as this by engaging some
suitable person to come in for an hour or two and stay with the
sleeping little ones.
All such details would have to be arranged according to personal and
local conditions; but the end to be attained is of such enormous
value that considerable effort is justified in reaching it. Even in the
beginning, a usefulness would be found in the united interest, the
mutual helpfulness of the combined women, drawn together by the
infinite and beautiful possibilities of their great work. In the light of
other eyes, they would see their own children in new lights, and, by
careful following of agreed lines of treatment, soon learn with some
finality what would and what would not be useful in a given case.
The observations and experiments of one earnest group of mothers
like this would be a stimulus and help to uncounted thousands of
ungrouped mothers who are struggling on alone.
It is by such effort as this, such interchange of view and combined
study, and the slowly accumulating record of established facts, that
humanity progresses in any line of similar work,—in floriculture or
horticulture or agriculture, or what you will; and this greatest of all
our labours, humaniculture, sadly lacks the application of the true
social law,—in union is strength.
The child needs not only love, but wisdom and justice; and these
grow best in the human soul through combination.
 XII.
MEDITATIONS ON THE NURSE-MAID.
"The trouble with these household problems which vex women so
much is that we do not give our minds to them sufficiently," said
earnest little Mrs. Blythe. "Now I mean to give my mind to this
nurse-maid problem, and work it out."
It is high time that somebody did. And it is not only on my own
account: this is something which affects us all,—all who have nurse-
maids, that is. I suppose the mothers without nurse-maids have
their problems, too; but I must consider mine now.
Now what is the matter with the nurse-maid? She does not suit me.
She has palpable faults and deficiencies. I want a better nurse-maid.
So far I have trusted to the law of supply and demand to produce
her, but it does not seem to work. I demand her, just as I have
demanded a better housemaid for some time; but the supply is not
forthcoming. So now I mean to think it out, and see if I cannot find
a way to the invention, discovery, or manufacture of a better nurse-
maid. And I mean to be very clear and logical in my thinking about
it, so as to come out in the end with proof. I want to prove what is
the matter with the nurse-maid and how to make her better.
In the first place, what are my objections to the nurse-maid now?
She is careless and irresponsible. She is ignorant. She is ill-
mannered. She is often deceitful. I can't trust her.
Now it doesn't seem right that my child should be placed in the care
of an ignorant, ill-mannered, careless, and irresponsible person,—
even if not also untrustworthy,—does it? And it does not relieve me
of the care as it ought. I have to take care of the child and the
nurse-maid, too. What I want is a careful, responsible, wise, well-
mannered, honourable young girl. She ought to have special
training, too. It is really dreadful the way these ignorant girls
undertake to care for children. We need schools—training schools—
and diplomas. They could have practice classes on the children of
the poor—or in institutions; and yet that idea does not quite suit me,
either. My child is very individual and peculiar, and I don't believe
that practising on poor children would fit a nurse-maid to take care
of my child. But nice people would not want their children to be
practised on. They would have to take the poor ones: it would do
them good, anyway. They get no care now: their mothers are
shockingly ignorant and neglectful.
But, after all, I don't have to arrange the training schools. I only
know that she ought to have special training, and it ought to be
practical as well as theoretical; and that means practising on some
children somewhere, somehow. And they certainly would have to be
poor, because rich people would not let their children go to be
practised on. Maybe the poor people would not, either. Then it would
have to be orphans, I guess, combining nurse-training schools with
orphan asylums, and foundlings, too.
Well, now these nurse-maids would go to these training schools to
improve themselves, would they! Come to think of it, they only go to
nursing because they need the pay; and, even if the training schools
were free, they'd have to wait longer for their money. And, if they
got no more with training than without, they would not go, I'm
afraid. We should certainly have to pay them more trained than
untrained. That is perfectly logical, I'm sure. And, of course, that
would be an obstacle. If the training schools were not free, we
should have to pay them more yet,—enough to make it worth while
to study the business of caring for children. A short course might do,
—six months or a year.
I've heard my mother say that she knew something about taking
care of children by the time Charley was born. But that was,—well, I
was eight, and I'm the third,—that was about twelve years. Oh, but
she wasn't in a training school! That would teach them faster. There
would be more children to practise on. Let me see: if it took my
mother twelve years to learn by practising on five children (Charley
was the fifth,—four children), how many children would it take to
learn on in one year? I'll get John to do that for me: I'm not good at
figures. Besides, it's different,—altogether different; for my mother
was a mother, so she knew how, to begin with, and nurse-maids are
not. So—to be strictly logical—it ought to take nurse-maids longer,
I'm afraid. The training schools will have to be free: I'm pretty sure
of that. And that means public or private endowment. We might as
well think it all out clearly.
Should it be added to the public-school system,—open to all girls,—
perhaps compulsory? Why not! Why wouldn't it be a good thing for
all girls to know something of the care of children? But could we do
that? Public schools are in politics; and that is awful. It would take
forever to get it that way; and my child wants a nurse-maid now!
Private endowment, I guess. So many rich people want to help the
masses. This would furnish employment, raise wages, and give us
nurse-maids. I'm sure it would appeal to any philanthropist.
Yes, some rich person must endow a training school for nurses,—
that sounds like hospitals; for child-nurses,—that sounds like wet-
nurses; for nurse-maids,—why need they be maids, though? Well, if
they were married, they would have children of their own of course,
and couldn't take care of ours. One would think, though, that
motherhood would give them more experience,—that they would
know how to care for children better. But, then, they wouldn't want
to leave their own children to take care of ours. And they couldn't
take care of them together. A mother would naturally do more for
her own: she wouldn't be fair.
A training school for nurse-maids. After all, "maid" does not mean
"unmarried" in this connection: it means simply "servant." And
"nurse" comes from the time when mere nursing was all that was
required,—a kind of a survival of old customs. How these things do
open up, when one thinks about them! Why "nurse-maid" at all!
Why not have a new and attractive name: that would help make
them go to the training school, too.
Nurse, nursing,—it isn't nursing our children want. They are not sick,
and they don't stay babies all the time they need this person. What
is it that our children need? Of course, they do need direct, personal
care; and, when they are babies, they need real "nursing,"—just
somebody to—to—well, they have to be fed,—and that only needs a
knowledge of infant physiology and nutrition; to keep the bottles
clean, of course, and be very accurate, and follow directions. They
don't need to know so much after all: the doctor tells what to give it
to eat and what not to. And the mother understands the child's
needs! Still, even for babies, they need some kind of training,—the
nurses, I mean,—not the mothers: it is divinely implanted in the
mother. And, then, mothers are studying these things now. I know
ever so many young mothers who are taking child-study now; and
about nutrition, too.
But the trouble is they can't depend on the nurses to carry out
instructions. If they were only trustworthy! Will the training schools
make them honourable? I suppose so. They would get some sense
of the importance and dignity of their work. They would be graded
and marked, of course, in their diplomas, so that one could pick out
the dependable ones; and that would gradually elevate the standard.
The trouble is, of course, when they go out. Children must be out of
doors; and, in cities where we have no yards, they cannot be under
the mother's eye, so they must be out with the nurse-maid. That's
perfectly logical. Then there are the other nurse-maids. One cannot
keep them isolated: that's out of the question. And if they have
admirers, as they do, of course,—young girls always will have
admirers, and training schools will not alter that,—why, if they meet
their admirers, it has a tendency to make them careless. That is
natural. We must allow for such things. And it is a perfectly natural
temptation to take the baby to see their own families. We forbid it,
of course; but I admit that it is a temptation. And there are all those
awful risks of diseases and things. Now, if their families were nicer
people and lived in nicer places,—but then they wouldn't want to be
nurse-maids! But if the training school raises wages and standards,
that will have an effect on the class of people who take up the work.
It certainly is the noblest, most beautiful, most important work in the
world,—the training of children. I wonder why our own girls do not
take it up,—our college girls. But then, of course, they wouldn't be
"nurse-maids." Perhaps, if it had another name—
Now let me think, and be fair. Would I want my sister Jessie to be a
nurse-maid? She is taking a kindergarten course, and we all approve
of that: it does help one so in all those problems that perplex a
mother! But, if she went to Mrs. MacAdoo's as a nurse-maid— The
MacAdoos are nice people, too; and the children are as nice as any I
know. They have a Swedish nurse-maid now,—a big, hearty,
wholesome-looking girl, but stupid. Why, she cannot answer the
simplest questions Harold asks, hardly; and he's always asking them.
Jessie has him in the kindergarten where she is. I don't mean that
she's the principal, but she is training there; and she tells me what a
bright child he is, and what stupid things Christine has told him. And
you see he has Jessie only three hours a day, and Christine all the
time he's awake. Jessie is taking a special course in infant
psychology, and she says Christine is doing him a world of harm. But
she is so good-natured and faithful that they keep her. They don't
realise that her being stupid is any harm to the children, I suppose.
But, if Jessie had him all the time, Harold certainly would develope
more rationally and more easily. And yet I am sure Jessie would not
take Christine's place. You see we visit the MacAdoos, and it would
be so awkward. Now, I think,—logically,—I am approaching a—I
forget the name of it, but it's a thing there's no way out of.
We would like our nurse-maids to be ladies, but ladies are not willing
to be nurse-maids. Now will the training school make ladies—or, at
least, partial ladies—of our nurse-maids? And, if it does, will that
make them disinclined to be nurse-maids? Or can we arrange the
position of the nurse-maid, so that ladies will be willing to take it?
What is the real difference between Jessie's position and Christine's?
Why, Jessie has a lot of children come to her part of the time; and
Christine has a few children, and goes to them all the time. And
Jessie has,—or will have when she's graduated and has a
kindergarten of her own, as I daresay she will,—she has control of
the children while they are with her, and can carry out her principles.
The mothers even consult her sometimes.
But Christine has to carry out the mother's orders. She does what
she is told—or ought to. No, Jessie never would be willing to take
Mrs. MacAdoo's orders about the children. Mrs. MacAdoo is
exceptionally stupid about children, I do think. She doesn't think
Christine's telling them stories about things to frighten them is any
harm,—says they'll outgrow it. And anybody who knows anything of
infant psychology knows how dangerous it is to frighten children.
And yet, of course, to be perfectly fair, I wouldn't want a nurse-maid
to dictate to me about my child. It is out of the question—absolutely.
Why, it would destroy the mother's influence and authority
altogether! And—come to think of it—I suppose a trained nurse-maid
would have views of her own, and they might conflict with the
mother's—
Now, where I have got to so far,—it is beautiful, thinking things out
clearly,—we want our children taken care of by ladies, honourable,
intelligent, educated, refined, and specially trained for the business.
I'm quite certain about that. Like Jessie, for instance. She is just
born for it,—always did love children, and knew how to manage
them from the time she was a little girl. And she's studying all the
science of it and practising in the kindergarten,—on the same kind of
children, too. Jessie is the ideal. It is really wonderful to see her with
them. They love her, and they do what she says, too; but she never
seems to be making them do anything: they just do it. Those
MacAdoos behave very much better with her than they do with their
mother. I believe most of the children do, for that matter. Except
little Cassie Wells. She has the most devoted mother I ever saw. It is
a lesson to us all. She never lets her out of her sight, I do believe.
Often comes to the kindergarten, just to be with her. And, you see,
Cassie just depends on her for everything; and nobody else can do
anything with her. It is beautiful,—such absolute dependence and
absorption. Yes, as I said, Jessie is the ideal. But, then, Jessie is not
a nurse-maid, and never would be.
Of course, if there was any way that Jessie could have the children
with her and have her way with them, as she does in the
kindergarten— But you can't do that with little children,—you cannot
separate the child from its mother! When they are older, they go to
school, of course; and, when they are older yet, they go to college,
and so on. But the little child needs its mother every hour. And, as
its mother cannot possibly give it every hour, we have to have the
nurse-maid. If mothers had no other claims, then, of course, you
would have the highest ideal relation. Cassie Wells's mother has
given up everything else. She doesn't go out with her husband at all.
Says that society has no claim beside that of the child. Of course, he
stays at home with her—mostly.
I'm sure a man ought to value his wife's society more than any
other, especially when she is such a devoted mother. She takes all
the periodicals about children, and reads all the books; and then she
modifies it all to suit her particular child. I never knew any mother
so conscientiously given up to the care of a child. She really talks of
nothing else. And, when that child is sick,—and she is extremely
delicate and always having dangerous illnesses,—her mother is
simply glued to her bedside: they can't drag her away. It is a pity
that the child is not better material; for she isn't particularly bright,
nor very well behaved, I think. But, then, her mother is doing
everything that can be done.
Jessie says that child is being mothered too much,—that she needs
more freedom and an impartial outside management. But, then,
Jessie is a good deal of a theorist; and, after all, she isn't a mother.
Nothing can really equal the mother's care for her own child! Still,
we simply can't do it,—all of us,—as families increase. We owe
something to our husbands, I am sure; and we have our social
duties; and our health is not always equal to such a strain. No, the
mother must have help; and that means the nurse-maid. It's no use
talking about Jessie. Even if she would do it, there's not enough of
her to go around! We never can expect that "faculty with children" in
everybody: they simply don't have it. Most girls don't care much for
children, nor know anything about them. Of course, after they
become mothers, it is different. Then it all comes to them.
Now, if nurse-maids could be mothers first— But I argued that out
before. If they were, they wouldn't be mothers of our children; and
motherhood only teaches how to do what is best for one's own
children. Besides, we couldn't hire them then, because we would not
separate mothers from their own children; and, if they had their
children and ours, too, they would not treat them fairly. And we
would not want them brought up with ours, either. No, they've got to
be "maids," that's sure.
Now the average young girl does not know or care much about
children. Therefore, she has to be trained. (What a comfort it is to
be really logical!) And, as there is no place to train them now, we
have got to make a place. It all comes round to the training school
for nurse-maids. That's the logical outcome.
Again, since we must have private nurse-maids under our orders,—
really a servant,—we cannot expect ladies to take such positions.
And—this ought to be bracketted with that last—since we cannot, of
course, pay more than so much, that is against ladies doing it, too.
Some people can, I know. Jessie told me of a very nice girl she
knew,—a classmate in college and a trained kindergartner,—who was
unable to get such a position as she wanted, and took a place with
some very rich people as a sort of lady nurse-teacher to the children.
But she said it was perfectly horrid, especially in travelling, having to
eat with servants and be treated as such. I can see that it would
take a kind of heroism, and we cannot really count on heroic nurse-
maids. No, it has to be from the lower classes that we take our
nurse-maids. I think that is proved. The average employer simply
couldn't pay them enough to attract a higher class of labour. These
are really questions of political economy in part, you see.
The ordinary young girl of the lower classes,—that is the raw
material of our nurse-maid. Naturally, she is ill-mannered or
unmannered, and careless and ignorant and all those things.
Therefore, we must train her. In order to do that, we must first
provide the training school, and, second, make her go to it. Now I
wonder how we could do that. The higher wages would be an object
of course: that would have to be insisted on. And we might "create a
sentiment." That's it! That's what we must do,—create a sentiment.
But it's no use doing anything till we've got the school. And I worked
that out as having to be done by private endowment. That involves
agitation, of course; and we must set about it. We can get teachers
plenty, there is so much interest in child-study now; and it will be a
splendid thing for the lower classes to take their young girls and
train them thoroughly in the theory of child-culture. It will make
them so much better mothers afterward, when they do marry, after
spending some years in taking care of our children,—putting their
theories in practice! But wait. That looks queer. Looks as if the rich
people were furnishing elaborate instruction free,—to young women
of the lower classes,—and then paying them good wages for
practising on the children of the upper classes, so that the poor
women might be better mothers afterward.
I must have made a mistake somewhere. I'm going to reverse that
position, and see how it would work. Suppose young girls of the
upper classes took elaborate instruction in child-culture, and then
practised on the children of the lower classes, in order to be better
mothers afterward. That seems more satisfactory, somehow; yet it
means a lot of work. It would do our girls good—I can see that—and
do the children of the lower classes good, and, no doubt, make the
girls better mothers. Besides, I'm wasting time,—"arguing in a
circle," John would say; for that upper-class-girl hypothesis wouldn't
give us nurse-maids. Now where was I? Mothers have to have help;
i.e., nurse-maids. These have to be private servants at low wages:
therefore, ladies would not do it. Therefore, we must have our
children taken care of by girls from the lower classes. They are not
suitable persons to take care of children as they stand: therefore, we
must train them.
Now I mean to really work for this thing,—to create a sentiment. I'll
begin early in the autumn, as soon as we get back. And I'm so glad
I'm going to have such a lovely summer to make me fit for it. You
see I'm very much pulled down. Little John has been such a care,
and the nurse-maids I've had have been so unreliable. Why, the
child has been sick again and again just through their carelessness.
I'm sure of it. And mother said I simply must go away and build up,
for the child's own sake; and John agreed with her—for once. And
there's such a lovely arrangement for the summer: nothing ever
happened more conveniently. You see Jessie is such an enthusiast
about children. And she has planned to be at home this summer. Our
home is perfectly lovely, anyway, and very healthy,—quite in the
country, and yet within easy reach of town. They're going to have
the Summer School of Child-study there at Seabay this year, and
Jessie has several of her class visiting her. And she said, in her
solemn, funny way, that they must have specimens to work on,—
first-class specimens! She insisted on little John, of course, and she's
persuaded Clara and George to let her have their three for a while;
and the little MacAdoos are to be there, too. It will be a regular
picnic for the children. It took a long time to bring me round to it.
But, then, it's my own lovely home. I know how healthy it is. And
mother will be there. And one of Jessie's friends is a doctor, and in a
children's hospital, too. She ought to see that everything is right for
their health. So, if they are happy in that lovely old place, and
healthy and well taught and safe, why, I suppose I can leave.
Of course, I wouldn't for anything on earth but health. Mrs. Wells
was perfectly horrified when I told her. They asked Cassie, too; but
she wouldn't hear of it. She said nothing but death should ever
separate her from her child. And, dear me, Cassie looked so white
that it really seemed as if it would. She made me feel guilty again;
but John can't come to any harm with my mother's experience and
Jessie's knowledge and natural talent. That's the main thing. Jessie
always cared more for children than I did,—except little John, of
course. They've fixed the place up on purpose for children. Such
arrangements for bathing and digging and mud-pieing and
gardening and so on you never saw. There is something for those
chicks to do all the blessed time, and these nice girls—my own
friends—to be with them every minute. You see they take turns and
relieve each other, so they are always fresh for the children. And,
then being so enthusiastic and scientific, it isn't drudgery to them.
They are studying all the time. And how glad I shall be to get back
in the fall! Then I can work up that training school for nurse-maids.
 XIII.
CHILDREN AND SERVANTS.
In the growing discontent with our present methods of household
service, while we waver between long-held prejudice, old and dear,
and the irresistible pressure of new conditions, it is worth while to
weigh well the relation between this present method of house-
service and our present method of child-culture.
The home is the place in which we rear young children. It is also the
place in which we perform certain kinds of labour, mainly cooking,
cleaning, and sewing. In the vast majority of our homes, fully nine-
tenths of them, as shown by the United States Census Report, giving
the number of domestic servants in proportion to the number of
families, these industries are carried on by the mother. She is the
domestic servant. In the remaining one-tenth of our homes the
labour is performed by hired servants, the maid-of-all-work still
greatly predominating. The questions here suggested for
consideration are: first, is a mother, who is also a house-servant,
able to supply proper conditions and care to young children? And,
second, is the company of domestic servants, other than their
mothers, and constant association with their industries, a desirable
condition for the education of young children?
It is, of course, difficult to consider with any clearness of perception
facts which have been always familiar. The association of child and
servant is so old that it makes no impression on our consciousness.
It will, perhaps, bring out the relation more vividly to change the sex
of the servant. Suppose a man is left with boys to educate. Suppose
he engages a tutor for his boys. He is willing to pay well for a man
with the proper ability, character, and training to come and benefit
his children by instruction and association. Would such a man be
willing to engage a tutor who was also a janitor? Would he be willing
to spare the time required to fill the janitor's position from the time
required to fill the tutor's position? Or would he be willing to engage
a man who had so little fitness for the profession of tutor as to be
content to act as janitor also?
Again, in sending his boys to school to be educated, would a man be
willing to have that school also run as a restaurant, a laundry, and a
tailor shop? Would he think these industries and the society of the
persons engaged in them good educational influences? It is clear
that a man would not be willing to do these things. Yet all men
cheerfully intrust their children, during their most impressionable
years, to the society and care of domestic servants and the constant
association with domestic industries. In most cases the servant is
also the mother. In other cases the servant is not the mother. In
either case the child grows up in association with domestic servants
and service.
Let us not too readily conclude that this is an evil, but examine it
carefully, in its physical and psychical effects. Physically, the child is
born into a certain kind of shop or factory. The conditions of any
labour in the home are particularly open to criticism; our sweat-shop
investigations show that in glaring instance. Intimate associations
with a trade, and especially a dirty or dangerous one, does not seem
advantageous to a child's health and progress. In nine homes out of
ten the child is directly associated with the trades of his mother, who
is a cook, a laundress, a cleaner in general; and the baby is early
accustomed to the fumes and heat of the kitchen, to grease and
ashes and dust, to all the kitchen-work, laundry-work, chamber-
work, and endless miscellaneous industries of his mother. In the
other tenth of our homes the child grows up a little removed, but
not far, from these same industries. They go on under his eyes none
the less, but with a certain ban upon them, as servant's work.
Any mother and housewife knows the complications continually
arising between children and servants. Early associations are deep
and lasting. Domestic servants are not, as a rule, either at all trained
in the right treatment of children or in such personal development of
character and manners as would make them desirable companions
for the young. Yet companions they are,—incessant, intimate,
unavoidable. The formative influence of a nurse-maid or of a maid-
of-all-work is of varying weight in different cases, but always a factor
in the child's development. The education of a child consists in every
impression received by the growing brain, not merely those received
when we are instructing it. We might give an hour a day to careful
instruction in good manners: we might ourselves be models of
propriety; but, if the child is also in the society of conspicuously ill-
mannered persons every day, an effect will surely be produced by
them.
It may be suggested that an end is to be attained through exhibiting
the deficiencies of servants, and exhorting the child to despise them,
as the Spartans used the Helots for an awful example; but, even if
this were gained, there would follow with it a spirit of scorn and
contempt for fellow-creatures most injurious to true social
development.
A little child should be surrounded with the best influences of all
sorts, and with behaviour not to avoid, but to imitate. The long
period of immaturity, which is one of our human distinctions, has its
value in the accumulated improvements which may be built into the
race in that time. It is a period of enrichment, of clear growth. To
expose the young to disadvantageous conditions, especially the very
young, is a method of education finding no precedent in nature and
no justification in reason. The adult, with developed powers, may
find in some degree of difficulty a stimulus to further effort; and, if
confronted with injurious conditions, may strive the harder to escape
or change them. But the new person, the child, has no background.
He can make no comparisons. He accepts his first environment
unquestioningly as "the world"; it is all the world he knows. For the
very reason that we were all born and reared in the domestic
factory, we find it hard to imagine any other conceivable
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