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METHODS IN MOLECULAR BIOLOGY™

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

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Lipoproteins and Cardiovascular
Disease

Methods and Protocols

Edited by

Lita A. Freeman
Cardiovascular & Pulmonary Branch, National Heart, Lung, and Blood Institute
National Institutes of Health (NIH), Bethesda, MD, USA
Editor
Lita A. Freeman
Cardiovascular & Pulmonary Branch
National Heart, Lung, and Blood Institute
National Institutes of Health (NIH)
Bethesda, MD, USA

ISSN 1064-3745 ISSN 1940-6029 (electronic)

ISBN 978-1-60327-368-8 ISBN 978-1-60327-369-5 (eBook)
DOI 10.1007/978-1-60327-369-5
Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2013938950

© Springer Science+Business Media, LLC 2013

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Preface

Methods in Molecular Biology: Lipoproteins and Cardiovascular Disease: Methods and Protocols
is a compendium of advanced and classical molecular biology methods targeted towards
lipoprotein, atherosclerosis, and vascular biology research.
Lipoprotein, atherosclerosis, and vascular biology studies present unique challenges to
the molecular biologist. The lipid-rich and otherwise challenging nature of many key
tissues complicate the isolation of high-quality RNA for gene expression analysis, for example,
and the unique nature of lipoproteins and their biological effects has engendered unique
methodologies. To date, no volume has yet encompassed these lipoprotein-centered cutting-
edge methods in molecular biology.
This book brings together in a single volume an updated set of protocols and strategies
for methods now driving advances in lipoprotein and atherosclerosis research, along with
classical methods that are still widely used. The chapters are written for researchers at any
level, from graduate students to established investigators with no prior experience in the
described techniques, and may be of interest to molecular biologists outside the lipoprotein
field using similar techniques.
Of particular interest to readers are methods chapters on quantitative real-time PCR,
microarrays, RT-PCR laser capture microdissection, and tissue-specific gene overexpres-
sion, knockout, and knockdown methodologies, including AAV as a liver-directed gene
delivery vehicle. Special topics include an overview of next-generation and third-generation
sequencing, antisense technology, chromatin immunoprecipitation, streamlined LCAT
activity assays, and native HDL subpopulation analysis. Updated methods for 5′ and 3′
RACE cloning of full-length cDNAs and Northern analysis have been added. Overviews,
strategic considerations, and background information are included for particularly novel or
complex methods.
This edition complements its classic predecessor, “Lipoprotein Protocols,” edited by
Jose Ordovas, by incorporating cutting-edge methodological advances developed over the
past decade. The two volumes together provide a complete, up-to-date set of methods for
any researcher with an interest in lipoproteins and their biological effects.
I would like to thank the following people for their contributions to this volume: John
Walker, the Series Editor, for his invaluable guidance and support, Gregory Kato, Robert
Shamburek, and my colleagues in the Pulmonary and Vascular Medicine Branch at NIH for
their support, patience, and encouragement, and Silvia Santamarina-Fojo and H. Bryan
Brewer for their guidance and contributions to lipoprotein metabolism over the years. My
former mentors and colleagues who taught me molecular biology over the years will find
their sage advice sprinkled throughout this volume—a small token of gratitude for their
efforts and encouragement. Many, many thanks as well to the Wolffe lab members. Finally,
this work would not have been accomplished without the bottomless support, encourage-
ment, and help from my friends, neighbors, and family.

Bethesda, MD, USA Lita A. Freeman

v
Contents

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

PART I RNA AND GENE EXPRESSION

1 Cloning Full-Length Transcripts and Transcript Variants

Using 5¢ and 3¢ RACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Lita A. Freeman
2 Monitoring Gene Expression: Quantitative Real-Time RT-PCR . . . . . . . . . . . 19
Elke M. Wagner
3 Microarray Technology: Basic Methodology and Application in
Clinical Research for Biomarker Discovery in Vascular Diseases . . . . . . . . . . . . 47
Nalini Raghavachari
4 Northern Analysis of Gene Expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Lita A. Freeman
5 Laser Capture Microdissection for Analysis of Macrophage Gene Expression
from Atherosclerotic Lesions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
Jonathan E. Feig and Edward A. Fisher

PART II SEQUENCING

6 Sequencing PCR-Amplified DNA in Lipoprotein and Cardiovascular

Disease Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
Victoria Youngblood and James G. Taylor VI
7 Introduction to Next-Generation Nucleic Acid Sequencing
in Cardiovascular Disease Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Lena Diaw, Victoria Youngblood, and James G. Taylor VI

PART III TRANSGENIC, KNOCKOUT, AND KNOCKDOWN METHODOLOGIES

8 Strategies for Designing Transgenic DNA Constructs . . . . . . . . . . . . . . . . . . . 183

Chengyu Liu
9 Purification of Plasmid and BAC Transgenic DNA Constructs. . . . . . . . . . . . . 203
Chengyu Liu, Yubin Du, Wen Xie, and Changyun Gui
10 Pronuclear Microinjection and Oviduct Transfer Procedures
for Transgenic Mouse Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Chengyu Liu, Wen Xie, Changyun Gui, and Yubin Du

vii
viii Contents

11 Genotyping of Transgenic Animals by Real-Time Quantitative PCR

with TaqMan Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Boris L. Vaisman
12 Generation of General and Tissue-Specific Gene Knockout
Mouse Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Xian-Cheng Jiang
13 Adeno-associated Viruses as Liver-Directed Gene Delivery Vehicles:
Focus on Lipoprotein Metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
William R. Lagor, Julie C. Johnston, Martin Lock, Luk H. Vandenberghe,
and Daniel J. Rader
14 Modulation of Lipoprotein Metabolism by Antisense Technology:
Preclinical Drug Discovery Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Rosanne M. Crooke and Mark J. Graham

PART IV SPECIAL TOPICS

15 Chromatin Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

Grant D. Barish and Rajenda K. Tangirala
16 Measurement of Lecithin–Cholesterol Acyltransferase Activity
with the Use of a Peptide-Proteoliposome Substrate . . . . . . . . . . . . . . . . . . . . 343
Boris L. Vaisman and Alan T. Remaley
17 Native–Native 2D Gel Electrophoresis for HDL Subpopulation Analysis . . . . . 353
Lita A. Freeman
18 Western Blots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
Lita A. Freeman

Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
Contributors

GRANT D. BARISH • Gene Expression Laboratory, Howard Hughes Medical Institute,

The Salk Institute for Biological Studies, La Jolla, CA, USA
ROSANNE M. CROOKE • Cardiovascular Disease Research, Antisense Drug Discovery,
Isis Pharmaceuticals, Carlsbad, CA, USA
LENA DIAW • Pulmonary and Vascular Medicine Branch, National Heart, Lung,
and Blood Institute, National Institutes of Health, Bethesda, MD, USA
YUBIN DU • iPSC and Genome Engineering Core, National Heart, Lung, and Blood
Institute, National Institutes of Health, Bethesda, MD, USA
JONATHAN E. FEIG • The Marc and Ruti Bell Vascular Biology Disease Program,
Department of Medicine (Cardiology), New York University School of Medicine,
New York, NY, USA; Department of Cell Biology, New York University School of
Medicine, New York, NY, USA
EDWARD A. FISHER • The Marc and Ruti Bell Vascular Biology Disease Program,
Department of Medicine (Cardiology), New York University School of Medicine,
New York, NY, USA; Department of Cell Biology, New York University School of Medicine,
New York, NY, USA
LITA A. FREEMAN • Cardiovascular & Pulmonary Branch, National Heart, Lung, and
Blood Institute, National Institutes of Health, Bethesda, MD, USA
MARK J. GRAHAM • Antisense Drug Discovery, Isis Pharmaceuticals, Carlsbad, CA, USA
CHANGYUN GUI • iPSC and Genome Engineering Core, National Heart, Lung, and Blood
Institute, National Institutes of Health, Bethesda, MD, USA
XIAN-CHENG JIANG • Department of Anatomy and Cell Biology, SUNY Downstate Medical
Center, Brooklyn, NY, USA
JULIE C. JOHNSTON • Penn Vector Core, Department of Pathology and Laboratory Medicine,
University of Pennsylvania School of Medicine, Philadelphia, PA, USA
WILLIAM R. LAGOR • Institute for Translational Medicine and Therapeutics, University of
Pennsylvania School of Medicine, Philadelphia, PA, USA; Cardiovascular Institute,
University of Pennsylvania School of Medicine, Philadelphia, PA, USA
CHENGYU LIU • iPSC and Genome Engineering Core, National Heart, Lung, and Blood
Institute, National Institutes of Health, Bethesda, MD, USA
MARTIN LOCK • Penn Vector Core, Department of Pathology and Laboratory Medicine,
University of Pennsylvania School of Medicine, Philadelphia, PA, USA
DANIEL J. RADER • Institute for Translational Medicine and Therapeutics,
University of Pennsylvania School of Medicine, Philadelphia, PA, USA;
Cardiovascular Institute, University of Pennsylvania School of Medicine, Philadelphia,
PA, USA
NALINI RAGHAVACHARI • Genetics and Developmental Biology, National Heart, Lung,
and Blood Institute, National Institutes of Health, Bethesda, MD, USA

ix
x Contributors

ALAN T. REMALEY • Lipoprotein Metabolism Section, Pulmonary and Vascular Medicine

Branch, National Heart, Lung, and Blood Institute, National Institutes of Health,
Bethesda, MD, USA
RAJENDA K. TANGIRALA • Division of Endocrinology, Diabetes, and Hypertension,
David Geffen School of Medicine, University of California Los Angeles,
Los Angeles, CA, USA
JAMES G. TAYLOR VI • Pulmonary and Vascular Medicine Branch, National Heart, Lung,
and Blood Institute, National Institutes of Health, Bethesda, MD, USA
BORIS L. VAISMAN • Lipoprotein Metabolism Section, Cardiovascular-Pulmonary Branch,
National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD,
USA
LUK H. VANDENBERGHE • Penn Vector Core, Department of Pathology and Laboratory
Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
ELKE M. WAGNER • Plasma Analytics/Development and Optimization, Baxter AG,
Wien, Austria
WEN XIE • iPSC and Genome Engineering Core, National Heart, Lung, and Blood Insti-
tute, National Institutes of Health, Bethesda, MD, USA
VICTORIA YOUNGBLOOD • Pulmonary and Vascular Medicine Branch, National Heart,
Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA
 Part I

RNA and Gene Expression

 Chapter 1

Cloning Full-Length Transcripts and Transcript Variants

Using 5¢ and 3¢ RACE
Lita A. Freeman

Abstract
Gene transcripts and transcript variants must be cloned to characterize gene function and regulation.
However, obtaining full-length cDNAs with accurate sequences from the 5¢ end through to the 3¢ end can
be challenging. Here we describe a reverse-transcriptase-based method for obtaining full-length cDNAs
using the SMARTer (“Switching Mechanism At RNA Termini”) RACE technology developed by Clontech.
RNA is isolated from the tissue of interest and annealed to a primer (a modified oligo(dT) primer for
polyA+ transcripts; random hexamers or a gene-specific primer for polyA− transcripts). A modified MMLV-
reverse transcriptase uses the primer to initiate cDNA synthesis from RNA transcript(s) annealed to the
primer and continues cDNA synthesis (reverse transcription) towards the 5¢ end of the transcript(s).
Importantly, this reverse transcriptase possesses terminal transferase activity, so when it reaches the 5¢ end
of a transcript it adds a 3–5 residue “tail” to the newly synthesized cDNA strand. Included in the reverse
transcriptase reaction mix is an oligonucleotide containing a sequence tag as well as a terminal series of
modified bases that anneal to the 3–5 residue tail on the newly synthesized cDNA. The reverse tran-
scriptase proceeds from the end of the transcript onwards into the modified bases and the rest of the
sequence-tagged oligo. The newly synthesized cDNA now has a sequence tag attached to it and can be
used as a template for PCR, with one primer complementary to the sequence tag and the second primer
specific to the gene of interest. The fragment can be cloned and sequenced or just sequenced directly. If
high-quality, undegraded RNA is used, obtaining the true 5¢ end of a transcript is greatly enhanced. In
combination with 3¢ RACE, full-length transcripts are easily cloned. This method provides sequence infor-
mation on important regulatory regions, such as 5¢ and 3¢ UTRs and flanking regions, and is ideal for
detecting transcript variants, including those with alternative transcriptional start sites, alternative splicing,
and/or alternative polyadenylation.

Key words RT-PCR, RNA, cDNA, RACE, 5¢ UTR, 3¢ UTR, mRNA, Noncoding RNA, Cloning,
Sequencing

1 Introduction

The central dogma of one gene, one transcript, one protein has
been transformed by systems biology. We now know that one
protein-encoding gene can produce multiple transcripts through a
variety of transcriptional or post-transcriptional processes, including
the use of alternative transcriptional start sites, alternative exon

Lita A. Freeman (ed.), Lipoproteins and Cardiovascular Disease: Methods and Protocols, Methods in Molecular Biology,
vol. 1027, DOI 10.1007/978-1-60327-369-5_1, © Springer Science+Business Media, LLC 2013

3
4 Lita A. Freeman

splicing, alternative splicing at short-distance tandem sites just a few

nucleotides apart, alternative polyadenylation, post-transcriptional
processing, and, rarely, RNA editing [1–9]. By definition, a protein-
encoding gene will have at least one of its transcripts translated into
protein. Alternative transcripts from the same gene, which share
some of the same exons as the major protein-coding transcript and
have protein-coding potential, may or may not be translated. These
alternative transcripts are often tissue-specific and their regulation is
not well understood. Protein-encoding genes can also contain inde-
pendent transcription units that do not share exons with the pro-
tein-coding transcripts. In fact, small RNAs (<200 nt) that do not
encode protein are transcribed not only from protein-coding genes
but also from intergenic regions [6]. Some are promoter-associated
small RNAs with an apparent role in transcription initiation [6, 10];
some are microRNAs with regulatory functions, for example miR-
122 [11], and the function of many small noncoding RNAs remains
to be determined [1, 2, 6]. Noncoding long RNAs with potential
regulatory roles in transcription have also been described [12].
Additional noncoding RNAs (ncRNAs) such as rRNAs, tRNAs,
telomerase RNA, and small nucleolar RNAs (snoRNAs) are well
characterized.
With the flood of information from systems biology, is cloning
and sequencing gene transcripts still necessary? For genes involved
in lipoprotein metabolism, the answer at this point is emphatically
yes. Proteomics and microarray analysis as well as exons genome and
RNA sequencing continue to reveal new potential therapeutic tar-
gets for atherosclerosis and dyslipidemia, and altered lipoproteins or
lipoprotein metabolism gene variants have been associated with dis-
eases as various as AIDS, pulmonary hypertension, kidney disor-
ders, and schizophrenia, as well as atherosclerosis. Moreover,
investigation of lipoprotein metabolism transcript variants has
uncovered unique regulatory mechanisms such as apoB mRNA
editing, and it is likely that additional co- or post-transcriptional
RNA processing mechanisms relevant to lipoprotein metabolism
remain to be discovered. To fully understand a protein-coding gene’s
regulation and function in lipoprotein metabolism and disease, all of
its transcripts and control mechanisms must be characterized, in all
relevant tissues. Only by cloning and sequencing of all of its tran-
scripts can a gene be fully characterized.
Most protein-encoding transcripts have a 5¢ cap, a 5¢ UTR, a
translation start site, a protein-coding sequence, a stop codon, a 3¢
UTR, and finally a 3¢ polyA tail (see Note 1). Cloning protein-coding
transcripts involves reverse transcription of RNA, starting from the
polyA tail, to form cDNA. Obtaining a full-length transcript and
determining the true 5¢ end of a transcript is not straightforward,
however. Strong secondary structure and/or high GC content within
the transcript can impede the progress of the reverse transcriptase,
which may fall off the transcript before reaching the true 5¢ end.
Incomplete 5¢ end sequence information can misidentify a downstream
Cloning Full-Length Transcripts and Transcript Variants Using 5¢ and 3¢ RACE 5

AUG as the N-terminus of the protein, leading to cloning and

sequencing of an incomplete protein lacking its correct N-terminus.
Incomplete 5¢ end sequence information will also lead to inaccurate
identification of the promoter and 5¢ UTR sequences. The 3¢ end of
the transcript must also be accurate to identify the C-terminus of the
protein and the 3¢ UTR sequence of the transcript, which can contain
regulatory signals (for example, see ref. 13). The true 3¢ end of a typi-
cal polyA+ mRNA transcript is not difficult to determine, since a long
run of A’s marks the end of the transcript. Obtaining the true 5¢ end
of protein-coding transcripts is generally the major hurdle.
RACE (“Rapid Amplification of cDNA Ends”), in which the
cDNA ends are sequence-tagged and then PCR-amplified using
primers complementary to the sequence tag or poly(dT) primer,
was an important advance in obtaining full-length transcripts.
However, even with improved reverse transcriptase technology, 5¢
RACE was still subject to incomplete reverse transcription and
inaccurate 5¢ end formation. We describe in this chapter a novel
technology, SMART (“Switching Mechanism At RNA Termini”)
RACE (now termed SMARTer RACE; see Note 2), that enriches
cDNA pools for 5¢ end sequences.
The 5¢ SMARTer RACE technology developed by Clontech is
specifically designed to amplify cDNA transcripts in which the
reverse transcriptase has reached a 5¢ end. The strategy of SMART
cDNA synthesis is shown in Fig. 1. PolyA+ RNA is annealed with
a modified oligo(dT) primer (“5¢-CDS primer A” for 5¢ RACE)
that binds the polyA+ tail (see Note 3). A modified MMLV-reverse
transcriptase (“SMARTScribe RT”) uses this primer to synthesize
cDNA from the transcript. Importantly, this particular reverse
transcriptase possesses terminal transferase activity, so when it
reaches the 5¢ end of the transcript it adds a 3–5 residue “tail” to
the newly synthesized cDNA strand (Fig. 1a). Included in the
reverse transcriptase reaction mix is the “SMARTer IIA
Oligonucleotide” which contains a sequence tag as well as a termi-
nal series of modified bases. These modified bases anneal to the
new 3–5 residue tail on the cDNA. The SMARTScribe RT is
tricked into believing that the SMART IIA oligo is contiguous
with the original RNA transcript and reverse-transcribes it. The
newly synthesized cDNA now has a sequence tag attached to it
(Fig. 1a) and can be used as a template for PCR, with one primer
complementary to the sequence tag and the second primer specific
to the gene of interest (Fig. 1b). High-temperature PCR increases
specificity and enhances processivity through difficult templates.
Suppression PCR combined with step-out PCR [14] provides
additional specificity. The resulting DNA fragments (see Note 4)
can be cloned and sequenced or just sequenced directly. If high-
quality, undegraded RNA is used, the odds of obtaining the true 5¢
end of a transcript are considerably enhanced. For 3¢ RACE a
modified oligo(dT) primer containing a 3¢ sequence tag (“3¢-CDS
primer A”) is used as a primer for reverse transcription with
6 Lita A. Freeman

SMARTScribe RT and a gene-specific primer can be used to PCR

the 3¢ end of the gene (Fig. 1b) (see Note 3). Conventional cloning
or end-to-end PCR is used to generate full-length cDNA (Fig. 1c).
The procedure described in this chapter has limitations. Very
strong secondary structure or GC-rich regions will interfere with
reverse transcription and RACE, regardless of the system. It is always

Fig. 1 Overview of SMART RACE. (a) Mechanism of SMARTer cDNA synthesis for 5¢ RACE. First-strand synthe-
sis is primed using a modified oligo(dT) primer. After SMARTScribe reverse transcriptase (RT) reaches the end
of the mRNA template, it adds several nontemplated residues. The SMARTer II A Oligonucleotide anneals to the
tail of the cDNA and serves as an extended template for SMARTScribe RT. The 5¢ RACE-ready cDNA, containing
extreme 5¢ end sequences of all polyA+ transcripts present in the RNA, is ready for PCR amplification of
specific genes. (b) Gene-specific primers used for 5¢ and 3¢ RACE amplification. Gene-specific primer 1 (GSP1)
is used for 5¢ RACE PCR and GSP2 is used for 3¢ RACE PCR amplification for a specific gene. Nested PCR using
nested gene-specific primers (NGSP) is necessary only when background or nonspecific amplification is high.
Further details are available in the manufacturer’s user manual. (c) Overview of steps involved in cloning full-
length cDNA using SMARTer RACE. 5¢ RACE generates 5¢ RACE fragments containing extreme 5¢ transcript
ends; 3¢ RACE generates 3¢ RACE fragments containing extreme 3¢ transcript ends. Options include cloning
and sequencing the entire 5¢ and 3¢ RACE fragments (recommended) or alternatively sequencing just the 5¢
end of the 5¢ product and the 3¢ end of the 3¢ product to obtain sequences of the extreme ends of the transcript.
This information is used to design 5¢ and 3¢ gene-specific primers to use in long-distance end-to-end PCR,
with the 5¢ RACE-ready cDNA as template, to generate the full-length cDNA. Overlap PCR is not recommended
(see Note 16). Full-length cDNA can also be obtained by conventional cloning of overlapping RACE fragments
if a suitable restriction site is present
 Cloning Full-Length Transcripts and Transcript Variants Using 5¢ and 3¢ RACE 7

Fig. 1 (continued)

a good idea to align 5¢ end sequences determined by 5¢ RACE

against the corresponding genomic sequence and inspect the
upstream 5¢ genomic sequence for GC-rich regions or predicted
strong secondary structure. 5¢ RACE can be repeated taking appro-
priate measures to overcome any GC-rich or strong secondary struc-
ture (see Note 5). Also, degraded RNA will present a 5¢ end that will
be sequence-tagged as if it were the true 5¢ end, so RNA degrada-
tion must be avoided at all cost. Finally, as far as we are aware the
SMARTer RACE procedure has not been used on small RNAs such
as microRNAs, which fare well with specialized linker-ligation-based
RACE methods [15, 16]. If the experimental goal is specifically to
sequence cDNA ends, especially in a high-throughput manner, with-
out cloning full-length cDNA, methods such as CAGE [17] or
Deep-RACE [18] should be considered. Chapters 6 and 7 of this
volume describe sequencing of PCR-amplified DNA and next-
generation (high-throughput) DNA sequencing methods [19, 20].
In general, however, the Clontech SMARTer RACE procedure
described below [14] is a simple and robust method that reliably
provides the most 5¢ sequence information, enabling cloning and
sequencing of full-length gene transcripts and their variants.
8 Lita A. Freeman

2 Materials

2.1 First-Strand 1. RNA isolation kit.

cDNA Synthesis Using 2. RNA quantitation and quality control reagents and equipment.
the SMART RACE
cDNA Amplification Kit
2.1.1 Before Beginning

2.1.2 First-Strand cDNA 1. Hot-lid thermal cycler (e.g., Gene Amp PCR System 9700
Synthesis thermal cycler, Applied Biosystems, Carlsbad, CA).
2. Thin-walled PCR tubes for thermal cycler.
3. SMARTer™ RACE cDNA Amplification Kit (Clontech
Laboratories, Mountain View, CA).
4. RNA isolated from tissue or cell type of interest.
5. (Optional) For polyA− transcripts: Poly(A) polymerase
(Catalog #2180A, Takara Bio, Madison, WI).
6. Molecular biology (MB)-grade, sterile water (certified RNase-
free, but not DEPC-treated).

2.2 RACE PCR Make sure the PCR is programmed in advance.

Reactions [14]
1. Molecular biology grade, sterile water (certified RNase-free,
but not DEPC-treated).
2. PCR polymerase, e.g., Advantage® 2 PCR Kit (Clontech),
Advantage® GC 2 PCR Kit (Clontech), Advantage® HF 2 PCR
Kit (Clontech), or other PCR system.
3. Thin-walled PCR tubes.
4. Hot-lid thermal cycler.
5. Gene-specific primers (see Note 6):
23–28 nt.
50–70 % GC.
Tm ³ 65 °C; for best results Tm > 70 °C.
Not complementary to the 3¢ end of the Clontech Universal
Primer.
Specific to gene of interest (check by BLAST).
6. If not using a hot-lid cycler, PCR-grade mineral oil (Sigma).
7. (Optional) For extreme RNA secondary structure: a thermo-
stable reverse transcriptase such as ThermoScript Reverse
Transcriptase (Invitrogen, Carlsbad, CA) and the GeneRACER™
kit (Invitrogen) with additional materials according to manu-
facturers’ instructions.
 Cloning Full-Length Transcripts and Transcript Variants Using 5¢ and 3¢ RACE 9

2.3 Cloning and 1. Agarose gel electrophoresis reagents and equipment.

Sequencing RACE 2. Ethidium bromide.
Products
3. Kit to extract DNA fragments from agarose gel slices for clon-
ing. For automated DNA purification (up to 12 samples) from
agarose gel slices in a spin-column format, the QIAcube from
Qiagen is convenient. For nonautomated DNA purification
from gel slices, the NucleoTrap Gel Extraction Kit Gel supplied
with the SMARTer RACE kit is a good choice, as is the QIAquick
or QIAEX Gel Extraction Kit (Qiagen, Valencia, CA).
4. TA-type cloning vector (for example the TOPO®-TA Cloning
Kit from Invitrogen). If the PCR products are blunt-ended,
use a ZeroBlunt® TOPO® cloning kit (Invitrogen) for cloning.
Use plates, liquid media, and other materials as recommended
by the supplier.

3 Methods

3.1 First-Strand 1. Isolate RNA from the tissue of interest. RNA used for 5¢ RACE
cDNA Synthesis Using or 3¢ RACE should be intact and free of DNA. The RNA isola-
the SMART RACE tion methods described in Chapters 2 and 3 of this volume
cDNA Amplification Kit [21, 22] are appropriate for 5¢ and 3¢ RACE as long as a
DNase-treatment step has been included. Other methods that
3.1.1 Before Beginning
produce RNA of similar quality are completely acceptable.
RNA integrity and concentration can be assessed as described
in Chapters 2 and 3 of this volume [21, 22].
2. Program your PCR machine with all constant-temperature and
cycling programs that will be used throughout the procedure.
3. Just before beginning the procedure, thaw needed reagents
on ice and keep cold (on ice). Keep the RNA on dry ice until
right before it is to be added to the reaction mix. Then thaw
it quickly by hand, place immediately on ice after thawing,
spin down briefly at 4 °C in a chilled, RNase-free microfuge,
add the RNA to the reverse-transcriptase tube, and refreeze
IMMEDIATELY in dry ice if it is to be reused (for something
other than RACE) at a later date. (Try not to reuse RNA that
has been thawed and re-frozen for RACE.)
4. Ensure that all procedures are performed in an RNase-free
manner and take great care not to cross-contaminate samples,
since PCR is used later in the procedure.

3.1.2 First-Strand cDNA 1. Preheat a hot-lid thermal cycler (e.g., Applied Biosystems Gene
Synthesis [14] Amp PCR System 9700 thermal cycler) to 70 °C.
2. Prepare Buffer Mix.
● For one 10 ml cDNA synthesis reaction, mix:
2.0 ml 5× First-Strand Buffer.
10 Lita A. Freeman

1.0 ml DTT (20 mM).

1.0 ml dNTP mix (10 mM).
Prepare enough for all the cDNA synthesis reactions, plus
one extra reaction (or 10 % extra), in a clean microtube. Spin
briefly in a microcentrifuge and then leave at room tempera-
ture until used in Subheading 3.1.2 step 8, below.
3. Combine in a separate 0.5 ml thin-walled PCR tube:
(a) For 5¢ RACE-ready cDNA:
● 1 – 2.75 ml total or polyA+ RNA (10 ng to 1 mg). For
the negative control, substitute an equivalent volume
of MB-grade water (see Notes 7 and 8).
● 1.0 ml 5¢-CDS primer A.
● Sterile MB-grade water to a final volume of 3.75 ml for
each reaction (see Note 8).
(b) For 3¢ RACE-ready cDNA:
● 1 – 3.75 ml total or polyA+ RNA (10 ng to 1 mg). For
the negative control, substitute an equivalent volume
of MB-grade water (see Notes 7 and 8).
● 1.0 ml 3¢-CDS primer A.
● Sterile MB-grade water to a final volume of 4.75 ml for
each reaction (see Note 8).
4. Mix contents and spin tubes briefly in a microcentrifuge.
5. Incubate the tubes at 72 °C in a hot-lid thermal cycler for
3 min, and then cool the tubes to 42 °C for 2 min.
6. Spin tubes briefly for 10 s at 14,000 × g in a microcentrifuge.
7. (FOR 5¢ RACE REACTION ONLY) Add 1.0 ml SMARTer
IIA oligo per reaction.
8. Prepare a Master Mix (enough for all cDNA synthesis reac-
tions, plus one extra reaction).
● To prepare Master Mix for one reaction, combine:
4.0 ml Buffer Mix (from step 2, above).
0.25 ml RNase inhibitor (40 U/ml).
1.0 ml SMARTScribe Reverse Transcriptase (100 U/ml).
Mix gently but thoroughly and centrifuge briefly to
collect contents at the bottom of the tube.
9. Add 5.25 ml Master Mix to each reaction tube containing
denatured RNA (step 6 for 3¢ RACE cDNA; step 7 for 5¢
RACE cDNA).
10. Mix the contents of each tube by gently pipetting and close top.
Spin briefly to collect the contents at the bottom of the tube.
 Cloning Full-Length Transcripts and Transcript Variants Using 5¢ and 3¢ RACE 11

11. Incubate the tubes at 42 °C for 1.5 h in a hot-lid thermal

cycler (or air incubator). Do not use a water bath.
12. Remove tubes from thermal cycler, and increase temperature
of thermal cycler to 70 °C.
13. Heat tubes at 70 °C for 10 min.
14. Dilute the first-strand reaction product with Tricine-EDTA
buffer:
Add 20 ml if you started with £200 ng of total RNA.
Add 100 ml if you started with ³200 ng of total RNA.
Add 250 ml if you started with polyA+ RNA.
15. Samples can be used immediately for 5¢ RACE and 3¢ RACE or
stored at −20 °C for up to 3 months (see Notes 9 and 10).

3.2 RACE PCR 1. Make sure the PCR is programmed in advance.

Reactions [14] 2. For each 50-ml PCR reaction, prepare a Master Mix:
34.5 ml PCR-grade water.
5 ml Advantage 2 PCR buffer.
1 ml dNTP mix (10 mM).
1 ml 50× Advantage 2 Polymerase mix (see Note 11).
Prepare enough for all the PCR reactions, plus one extra
reaction (or 10 % extra), in a clean microtube. Mix well to
vortex, avoiding bubble formation, and spin briefly.
(a) FOR 5¢ RACE:
To each 0.5 ml PCR tube add (in the order shown):
2.5 ml 5¢ RACE-ready cDNA from Subheading 3.1.2 step 14.
5.0 ml UPM.
1.0 ml GSP1 (10 mM).
41.5 ml Master Mix.
(b) FOR 3¢ RACE:
To each 0.5 ml PCR tube add (in the order shown):
2.5 ml 3¢ RACE-ready cDNA from Subheading 3.1.2 step 14.
5.0 ml UPM.
1.0 ml GSP2 (10 mM).
41.5 ml Master Mix.
The final volume will be 50 ml per tube.
3. Mix gently.
4. Centrifuge briefly (in a PCR minifuge if available).
5. Begin PCR using the appropriate program and a hot-lid ther-
mal cycler.
12 Lita A. Freeman

(NOTE: If not using a hot-lid cycler, overlay contents with two

drops of mineral oil.)
For Program 1 (if GSP Tm > 70 °C) (RECOMMENDED)
5 Cycles:
94 °C 30 s.
72 °C 3 min (if the expected fragment will be >3 kb, add
1 min for each additional 1 kb).
5 Cycles:
94 °C 30 s.
70 °C 30 s.
72 °C 3 min (if the expected fragment will be >3 kb, add
1 min for each additional 1 kb).
20 Cycles (polyA+) or 25 cycles (total RNA).
94 °C 30 s.
68 °C 30 s.
72 °C 3 min (if the expected fragment will be >3 kb, add
1 min for each additional 1 kb).
NOTE: If the GSP Tm = 60–70 °C, use:
20 Cycles (polyA+ RNA) or 25 cycles (total RNA):
94 °C 30 s.
68 °C 30 s.
72 °C 3 min (if the expected fragment will be >3 kb, add
1 min for each additional 1 kb).

3.3 Cloning and 1. Analyze by gel electrophoresis, stain with ethidium bromide
Sequencing RACE (see Note 12), and excise all bands (see Notes 13 and 14).
Products 2. Isolate DNA fragments from the gel slices using your preferred
gel extraction method. For automated DNA purification (up to
12 samples) from agarose gel slices in a spin-column format, the
QIAcube from Qiagen is convenient. For non-automated DNA
purification from gel slices, the NucleoTrap Gel Extraction Kit
Gel supplied with the SMARTer RACE kit is a good choice, as
is the QIAquick or QIAEX Gel Extraction Kit (Qiagen).
3. Clone the gel-purified fragments into a TA-type cloning vector
(for example using a TOPO®-TA Cloning Kit from Invitrogen).
If you used a PCR polymerase that gives blunt-ended PCR
products, use a ZeroBlunt® TOPO® cloning kit for cloning the
PCR product.
4. To obtain the maximum amount of 5¢ end sequence, sequence
8–10 clones from EACH fragment excised from the gel. Some
genes have multiple initiation sites over a ~100 bp region rather
than one discrete transcription initiation site. If this appears to
be the case, sequence more clones to ensure the 5¢ end has
really been reached (see Note 15).
 Cloning Full-Length Transcripts and Transcript Variants Using 5¢ and 3¢ RACE 13

5. To obtain a full-length clone, perform long-distance PCR with

a high-fidelity polymerase using primers from the extreme 5¢
and 3¢ ends and the 5¢ RACE-ready cDNA (Subheading 3.1.2
step 14) (see Note 16).
6. Several databases provide sequence information on gene tran-
scripts and transcript variants, either predicted in silico or experi-
mentally verified. (See ref. 23 for a recent list of transcript
databases. New databases are likely to emerge, so check the lit-
erature.) It is always a good idea to check your sequence results
against existing databases and/or previously published papers,
whether you are seeking confirmation that you have the “right”
sequence or whether you are looking for novel variants.

4 Notes

1. A notable exception is the class of replication-dependent his-

tone mRNAs, which encode protein but lack a polyA tail. Also
note that while protein-coding transcripts are generally capped,
a transcript with a 5¢ cap will not necessarily be translated into
protein. That is, many noncoding RNAs are capped [6].
2. The original SMART RACE cDNA Amplification Kit was
replaced in February 2009 by a new, improved version termed
the SMARTer RACE cDNA Amplification Kit. The principles
behind the procedure have not changed but some experimen-
tal details have. For example, the newer kit uses a new reverse
transcriptase and modified oligonucleotide sequences, and less
RNA can be used in the RT step. If you refer to an online pro-
cedure when using the kit, make sure it’s the updated protocol
for the SMARTer RACE kit.
3. Many noncoding RNAs lack a polyA tail or are “poorly poly-
adenylated” [9]. To amplify transcripts that lack a poly(A) tail,
a poly(A) tail can be added to the 3¢ end of the transcript using
Poly(A) polymerase (Takara). SMARTer RACE can then be
carried out as usual, using 5¢-CDS or 3¢-CDS primer A to
prime the reverse transcriptase reaction for 5¢ or 3¢ RACE,
respectively. Traditional linker-ligation RACE can also be used
to determine the 5¢ and 3¢ ends of polyA− transcripts. If you
don’t need 3¢ end information and just need the 5¢ end of a
polyA− transcript, use the SMARTer RACE kit with random
primers or a primer of known sequence to prime the reverse
transcriptase reaction.
4. Usually multiple bands representing different transcript variants
are apparent on an agarose gel after RACE. All bands should be
excised from the gel and cloned and sequenced. It is sometimes
assumed that the longest 5¢ RACE product size contains the
14 Lita A. Freeman

“real” 5¢ end and that smaller 5¢ RACE products are merely

incomplete reverse-transcription products, but this is not a valid
assumption. Transcript heterogeneity is commonplace.
5. For truly intractable RNA secondary structure, reverse tran-
scription should be performed at higher temperatures using
a thermostable reverse transcriptase such as ThermoScript
Reverse Transcriptase (Invitrogen). ThermoScript RT can
generate cDNA transcripts from 100 bp to >12 kb at tem-
peratures ranging from 50 to 65 °C. Higher temperatures
will promote denaturation of secondary structure. This
enzyme can be used in Invitrogen’s GeneRACER™ kit
according to manufacturers’ instructions. Somewhat to our
surprise, we have found that kits designed to specifically
amplify transcripts with a 5¢ cap do not necessarily yield
more 5¢ sequences than the SMARTer RACE kit, perhaps
because of the increased handling required to capture the
capped transcripts. Ease of use, selection for full-length
cDNAs, and high specificity have led to our preference for
SMARTer RACE technology. Efficient PCR amplification of
GC-rich regions or sequences with strong secondary struc-
ture is a separate issue that is easily remedied by adding a
cosolvent or increasing the temperature during the PCR
step (see Note 11, below).
6. The recommended length of 23–28 nt refers to the length of
overlap with the target sequence. Additional nucleotides, for
example restriction sites for cloning, can be placed on the 5¢
ends of the oligonucleotide. If the PCR product is to be
restriction-enzyme digested and cloned directly into the vec-
tor of interest without prior subcloning into a TA-type vector
(see Subheading 3.3), an additional 6–8 nt of random sequence
should be added 5¢ of the restriction site to ensure complete
digestion by the restriction enzyme. If possible, use the same
nucleotides in opposite directions for 5¢ and 3¢ RACE to mini-
mize formation of a chimeric DNA fragment after PCR.
7. Negative and positive controls: It is essential to include a nega-
tive control (no RNA added) every time a reverse-transcription
reaction is performed to check for cross-contamination. The
positive control included in the kit (control human placental
DNA) is also highly recommended, especially for the beginner,
to be used according to manufacturer’s instructions. As an
additional positive control to test cDNA made from your own
RNA sample, use the cDNA created from in this step from
your sample RNA to perform RACE for a gene whose 5¢ end
has already been determined.
8. Do not use DEPC-treated water. Residual DEPC may inhibit
the reverse transcriptase.
Cloning Full-Length Transcripts and Transcript Variants Using 5¢ and 3¢ RACE 15

9. Samples can be stored long-term at −70 °C. Avoid repeated

freeze-thaw cycles.
10. The samples can be saved and used for other applications that
use cDNA as a starting material.
11. For efficient amplification through GC regions, Clontech rec-
ommends the Advantage GC 2 PCR kit (#633119 & #639120).
Alternatively, a cosolvent additive (e.g., PCRx Enhancer,
Invitrogen, Carlsbad, CA) [24] that helps to “melt” DNA can
be added to the PCR reaction according to manufacturer’s
instructions; optimization may be required. For highest fidelity
Clontech recommends the Advantage HF 2 PCR kit (#639123
& #639124). We suggest using either the Advantage 2 PCR or
Advantage GC 2 PCR kits for the initial 5¢ RACE or 3¢ RACE
PCR amplifications (and for nested PCR if necessary), since
the SMARTer RACE kit has been optimized using these
reagents. Once the extreme 5¢ and 3¢ ends are known, the user
can use the Advantage HF 2 PCR kit, or alternatively the high-
fidelity PCR enzyme of their choice, along with primers cor-
responding to the 5¢ and 3¢ ends, to amplify full-length
fragments from cDNA for cloning and/or sequencing.
12. If no bands are visible on an ethidium bromide-stained agarose
gel, perform nested PCR using the Nested Universal Primer A
(NUP; 10 mM), which is included in the SMARTer RACE kit,
and a second gene-specific primer.
13. Cut out all bands, not just the band with the highest molecular
weight, and clone and sequence them. Multiple bands result
from alternative transcription initiation sites, alternative splic-
ing, and alternate polyadenylation events, and it cannot be
assumed that the highest-MW band, or even the most intense
band, corresponds to the major transcript that is translated
into the major protein isoform. Cloning and sequencing alter-
native transcript variants as well as the primary transcript can
frequently yield new insights into gene function and regula-
tion. Just as frequently, rather bizarre transcript variants, lack-
ing any apparent function, turn up as well. Such transcripts are
regarded as valid, albeit mysterious, and should not cause
alarm. Importantly, if the number of bands seems excessive,
ensure that the RNA is not degraded and troubleshoot the
procedure as described in the SMARTer RACE cDNA
Amplification Kit User Manual.
14. Shotgun cloning of 5¢ RACE or 3¢ RACE reactions is not rec-
ommended since shorter clones will be overrepresented.
15. As a check, alternative RACE methods (see Note 5) or 5¢
CAGE [17, 25] using a gene-specific primer or other method-
ologies that provide RNA sequences adjacent to the 5¢ cap can
also be investigated. Note, however, that 5¢ CAGE sequences
16 Lita A. Freeman

are not always promoter adjacent [1, 6]. Chromatin IP with an

antibody specific for RNA Polymerase II phosphorylated at Ser
5 can be used to verify transcription initiation within a ~300–
500 bp region [26], and promoter activity can be tested directly
by cloning the promoter in front of a luciferase gene, for exam-
ple using one of the pGL4 vectors from Promega, transfecting
into the appropriate cell type and assaying luciferase activity.
16. Although some protocols suggest using overlap PCR between
the 5¢ and 3¢ RACE products to obtain the full-length PCR
product, this method introduces the possibility that two tran-
scripts with closely related but nonidentical overlap sequences
may co-amplify to form a chimeric transcript. It is better to use
the 5¢ and 3¢ RACE sequence information to design primers and
then perform 5¢ end-to-3¢ end PCR with a high-fidelity PCR
enzyme from the cDNA prepared in Subheading 3.1.2 step 14.
The full-length fragment is then cloned and sequenced.

References
1. Gustincich S, Sandelin A, Plessy C, Katayama S, 10. Core LJ, Waterfall JJ, Lis JT (2008) Nascent
Simone R, Lazarevic D, Hayashizaki Y, Carninci RNA sequencing reveals widespread pausing
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the transcriptional landscape. Mamm Genome SK, Pear M, Watts L, Booten SL, Graham M,
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Davuluri RV (2008) Genome-wide analysis of (2006) miR-122 regulation of lipid metabo-
alternative promoters of human genes using a lism revealed by in vivo antisense targeting.
custom promoter tiling array. BMC Genomics Cell Metab 3:87–98
9:349 12. Carninci P (2009) Molecular biology: the long
4. Kim E, Goren A, Ast G (2008) Alternative splic- and short of RNAs. Nature 457:974–975
ing: current perspectives. Bioessays 30:38–47 13. Hao S, Baltimore D (2009) The stability of
5. Hiller M, Platzer M (2008) Widespread and mRNA influences the temporal order of the
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tandem sites. Trends Genet 24:246–255 molecules. Nat Immunol 10:281–288
6. Affymetrix/CSHL ENCODE Project (2009) 14. Clontech (2009) SMARTer RACE cDNA
Post-transcriptional processing generates a amplification kit user manual. Clontech,
diversity of 5¢-modified long and short RNAs. Mountain View, CA, pp 1–33
Nature 457:1028–1032 15. Kong W, Zhao JJ, He L, Cheng JQ (2009)
7. Powell LM, Wallis SC, Pease RJ, Edwards YH, Strategies for profiling microRNA expression.
Knott TJ, Scott J (1987) A novel form of J Cell Physiol 218:22–25
tissue-specific RNA processing produces apoli- 16. Sdassi N, Silveri L, Laubier J, Tilly G, Costa J,
poprotein-B48 in intestine. Cell 50:831–840 Layani S, Vilotte JL, Le PF (2009) Identification
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BR, Weng SA, Silberman SR, Cai SJ, Deslypere from normal mouse mammary gland. BMC
JP, Rosseneu M (1987) Apolipoprotein B-48 Genomics 10:149
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organ-specific in-frame stop codon. Science M, Fukuda S, Tagami M, Sasaki D, Imamura
238:363–366 K, Kai C, Harbers M, Hayashizaki Y, Carninci
9. Carninci P (2006) Tagging mammalian tran- P (2006) CAGE: cap analysis of gene expres-
scription complexity. Trends Genet 22:501–510 sion. Nat Methods 3:211–222
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18. Olivarius S, Plessy C, Carninci P (2009) High- 23. Koscielny G, Le TV, Gopalakrishnan C,
throughput verification of transcriptional start- Kumanduri V, Riethoven JJ, Nardone F,
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19. Youngblood V, Taylor JV (2010) Sequencing F, Ritchie W, Moucadel V, Ara T, Pospisil H,
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(ed.) Methods in molecular biology: lipopro- Thanaraj TA, Gautheret D (2009) ASTD: the
teins, 2nd edn, Walker JM (series ed.). alternative splicing and transcript diversity
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 Chapter 2

Monitoring Gene Expression: Quantitative

Real-Time RT-PCR
Elke M. Wagner

Abstract
Two-step quantitative real-time RT-PCR (RT-qPCR), also known as real-time RT-PCR, kinetic RT-PCR,
or quantitative fluorescent RT-PCR, has become the method of choice for gene expression analysis during
the last few years. It is a fast and convenient PCR method that combines traditional RT-PCR with the
phenomenon of fluorescence resonance energy transfer (FRET) using fluorogenic primers. The detection
of changes in fluorescence intensity during the reaction enables the user to follow the PCR reaction in
real time.
RT-qPCR comprises several steps: (1) RNA is isolated from target tissue/cells; (2) mRNA is reverse-
transcribed to cDNA; (3) modified gene-specific PCR primers are used to amplify a segment of the cDNA
of interest, following the reaction in real time; and (4) the initial concentration of the selected transcript in
a specific tissue or cell type is calculated from the exponential phase of the reaction. Relative quantification
or absolute quantification compared to standards that are run in parallel can be performed.
This chapter describes the entire procedure from isolation of total RNA from liver and fatty tissues/
cells to the use of RT-qPCR to study gene expression in these tissues. We perform relative quantification
of transcripts to calculate the fold-difference of a certain mRNA level between different samples. In addi-
tion, tips for choosing primers and performing analyses are provided to help the beginner in understanding
the technique.

Key words RT-PCR, qPCR, RNA extraction, TaqMan® probe, Endogenous control, Liver,
Macrophages, Relative quantification, Single-well reaction

1 Introduction

Lipoproteins have a vital role in transporting hydrophobic lipids

through the aqueous milieu of the circulation. Triglycerides, cho-
lesterol (free cholesterol and cholesteryl esters), and phospholipids
are the major lipid constituents of lipoproteins, while other small
hydrophobic molecules such as fat-soluble vitamins or lipophilic
drugs may be carried in lipoproteins as well [1–4].
Given their critical roles in supplying hydrophobic molecules
to cells and tissues as well as in preventing accumulation of excess
lipid throughout the body, it is not surprising that genes involved

Lita A. Freeman (ed.), Lipoproteins and Cardiovascular Disease: Methods and Protocols, Methods in Molecular Biology,
vol. 1027, DOI 10.1007/978-1-60327-369-5_2, © Springer Science+Business Media, LLC 2013

19
20 Elke M. Wagner

in lipoprotein metabolism are highly regulated. Gene variants,

gene under- or over-expression (for example in transgenic or
knockout mice), dietary influences, and therapeutic agents that
alter lipoprotein metabolism can change gene expression patterns
in many cells and tissues: liver and intestine, which produce apoB-
containing lipoproteins and HDLs; macrophages, endothelial cells,
inflammatory cells, and smooth muscle cells in the artery wall; and
other cell types such as adipose tissue, lungs, pancreatic b-cells, and
cytokine/chemokine-secreting cells. In addition, the brain has its
own separate lipoprotein metabolism due to the inability of lipids/
lipoproteins to cross the blood–brain barrier [5]. As different tis-
sues may regulate the same gene quite differently, it can be neces-
sary to characterize expression of the same gene(s) in multiple
tissues. Clearly, a fast, high-throughput, cost-effective, quantita-
tive, and reproducible method for measuring gene expression, such
as quantitative real-time PCR (qPCR)—specifically, qPCR in com-
bination with reverse transcription (RT-qPCR) [6–8]—will be
advantageous to any state-of-the-art laboratory investigating lipo-
protein metabolism.
RT-qPCR, also known as real-time RT-PCR, kinetic RT-PCR,
or quantitative fluorescent RT-PCR, combines traditional
RT-PCR [9] with the phenomenon of fluorescence resonance
energy transfer (FRET) using fluorogenic primers [10]. While
RT-qPCR is not without limitations, it can provide unmatched
sensitivity and speed for determining levels of specific transcripts
when using proper controls, careful initial reaction characteriza-
tion, and quality control.
In order to measure gene expression by RT-qPCR, RNA is
isolated and reverse-transcribed using random hexamers as primers
to form cDNA (see Note 1). Gene-specific PCR primers are then
used to amplify a segment of the cDNA of interest. The reaction is
followed in real time by detecting fluorogenic tags that are cova-
lently linked to modified primer(s); alterations in fluorescence
intensity during each PCR reaction cycle enables the user to follow
the PCR reaction in real time. Several fluorogenic PCR assays with
a variety of innovative primer designs are available to date (TaqMan®
hydrolysis probes, Molecular Beacons, LUX primers, hybridization
probes, Scorpion primers, Sunrise primers, etc.); a good overview
for common qPCR assays can be found in Wong and Medrano
[10]. For the purpose of this basic protocol, the TaqMan® hydro-
lysis probe (ABI) is described (see Fig. 1a).
The TaqMan® probe is a linear, dual-labeled oligonucleotide of
20–30 nucleotides (nts) that anneals to the sequence between the
two traditional PCR primers. It contains a fluorogenic reporter dye
on its 5¢ end and a quencher molecule on its 3¢ end (Fig. 1a). The
reporter dye is hydrolyzed by the 5¢ nuclease activity of the PCR
enzyme during each new strand synthesis and emits fluorescence
once it is liberated and out of range of the quencher dye on the
 Monitoring Gene Expression: Quantitative Real-Time RT-PCR 21

Fig. 1 Principle of qPCR using a TaqMan® probe. (a) Schematic of a qPCR reaction. In addition to the traditional
two PCR primers, a third primer with two covalently linked tags on its 5¢ and 3¢ end anneals to the DNA. When
the 5¢ fluorogenic reporter dye (e.g., “FAM,” “VIC,” or other dyes) is excited by light, its emission will be
quenched while bound to the primer and in close proximity to the nonfluorescent quencher molecule on the 3¢
end. As the 5¢ exonuclease activity of the Taq DNA polymerase hydrolyzes the 5¢ reporter dye during strand
extension, the dye is liberated and emits fluorescent light. The amplification can thus be followed using a
fluorescent light detector. The most basic outfit of a real-time instrument would consist of a PCR running in
optical reaction tubes on a thermo cycler with a lamp (excitation) and a detector (fluorescence). (b) Data analy-
sis of TaqMan® assay using the 7300 SDS software. Upper panel: Linear view of a real-time PCR amplification
curve. Lower panel: Logarithmic view of the same curve. The exponential phase seems now “linearized.”
During this exponential increase, we analyze the qPCR, reading the number of cycles at a chosen “threshold”
reporter signal intensity (Delta Rn). The cycle at threshold (Ct) for the curve is 20.5. It can be compared to other
samples at the same threshold; relative gene expression levels between, for example, a wild-type and a trans-
genic organism, can easily be derived from the number of PCR cycles needed to achieve the same quantity of
fluorescent light (“threshold”). Rn, measure of fluorescent reporter signal. Delta Rn, measure of fluorescent
reporter signal corrected for baseline/background

probe. The rise in fluorescence intensity is detected and presented

graphically as light intensity (Delta Rn) vs. PCR cycles (cycle num-
ber) (see Fig. 1b). The use of three gene-specific oligonucleotides
in the TaqMan® assay assures highly specific amplification of even
short cDNA sequences (typically 60–100 nts).
When studying gene expression, “relative” quantification is
used to compare the gene expression levels between different
experimental conditions or tissues. In this case, differences are
expressed as “fold-changes.” Absolute quantification of DNA copy
numbers may be of interest for the determination of viral loads or
22 Elke M. Wagner

transgene integration (see Chapter 11 of this volume [11]). For the

analysis of a qPCR experiment, a “threshold” level of fluorescence
is chosen in the exponential phase of the PCR, and the number of
cycles required for fluorescence to reach this threshold level [termed
cycle at threshold (Ct) or cycle at crossing point (Cp)] is determined
(see Fig. 1b). Relative amounts of copy numbers of a transcript in
different samples can be assessed by comparing their Cts, with
smaller Cts corresponding to higher initial copy numbers [12]. In
order to assure that the same amount of total cDNA is compared,
a loading control (or “housekeeping gene”) is detected in addition
to the gene of interest in each sample.
In qPCR, the ability to follow and analyze the reaction prog-
ress during the early exponential phase minimizes confounding
factors that affect late cycles (e.g., in the end-point quantification
of traditional PCR), such as reagent consumption, slower reac-
tions, and reaction product degradation [13]. This improves the
reproducibility and accuracy of the initial transcript copy number,
and reduces hands-on steps and hence the possibility of contami-
nation. However, it is important to note that RNA species are not
directly visualized by this method and that unspecific reactions,
isoforms, and splice variants will go undetected.
The following chapter describes the isolation of total RNA of
high quality from liver and fatty tissues/cells and the use of
TaqMan® Gene Expression Assays to study gene expression in these
tissues (see Note 2). Tips for choosing primers are provided to help
the first-time user in understanding the technique. We have
included a detailed description of manual data analysis for perform-
ing relative quantification with a standard curve. While this may
appear laborious compared to automated software modules, we
feel this leads to a basic understanding of the methodology and will
help to estimate the results and to keep the user informed of hid-
den biases in automated analysis.
We encourage the reader to inform himself/herself about PCR
and qPCR as well as the instrument/software to be used. Very
concise and useful brochures are accessible for all users on the ABI
home page [12–14]. Finally, we discuss potential pitfalls for the
reader to keep in mind when setting up a specific assay for a certain
tissue/cell line for the first time.

2 Materials

If materials change, make sure to get comparable material or con-

tact provider and ask for substitute products. We assume that
pipettes, RNase- and DNase-free pipette tips (filtered), centrifuges,
agarose gel apparatus, and gloves are available in the lab.
 Monitoring Gene Expression: Quantitative Real-Time RT-PCR 23

2.1 Total RNA IMPORTANT: Wear clean gloves while preparing all reagents and
Extraction from Tissue equipment for RNA extraction and during the extraction. Change
Using TRIzol gloves as needed. The workspace and all tools listed below should
be wiped with RNaseZap and/or 70 % ethanol. Purchase several
liters of RNase-free water (Ambion Inc., Austin, TX), absolute
ethanol (Sigma-Aldrich, St. Louis, MO) and prepare 70 % wash
alcohol with RNase-free water.

2.1.1 Isolation and 1. Tissue/paper towels.

Stabilization of Tissue 2. RNaseZap (Ambion Inc., Austin, TX).
3. 70 % Ethanol, prepared with RNase-free water in a wash
bottle.
4. RNAlater (Ambion Inc.) or equivalent (e.g., Allprotect
Reagent, Qiagen, Valencia, CA).
5. Optional: a Styrofoam lid and pins to hold the mouse corpse in
place.
6. Two pairs of scissors, wiped clean.
7. Two blunt forceps and two bent, blunt forceps, wiped clean.
8. Small glass plate (about 10 cm × 10 cm), wiped clean.
9. RNase-free tubes, 2 ml (e.g., Safe-Lock Eppendorf; Fisher
Scientific, Pittsburgh, PA). Use with clean gloves. Dedicate a
box or bag of tubes for work with RNA only. Keep bag
closed.
10. Liquid nitrogen or dry ice/ethanol bath for snap-freezing the
tissues after excision.

2.1.2 RNA Extraction 1. 50 ml BD/Falcon tubes (Fisher Healthcare, Pittsburgh, PA).

from Tissue (TRIzol) 2. Polytron/Ultra-Turrax [Kinematica Inc., Bohemia, NY
(formerly Brinkmann Instruments)] or equivalent homog-
enizer, rinsed with RNaseZap or 70 % alcohol (in 50-ml
tubes). Wipe dry.
3. Aliquot 30 ml of RNase-free water into 50 ml tubes (one per
sample, used to rinse homogenizer).
4. TRIzol reagent (Invitrogen, Carlsbad, CA): avoid skin con-
tact, wear coat and gloves, keep in refrigerator, and always
clean the outside of the bottle well after use. Use appropriate
thick-walled waste containers, as organic compounds dissolve
some plastic containers (and plastic bags) readily!
5. Centrifuge tubes appropriate to the centrifuge available: e.g.,
for the SA-600 rotor on a Sorvall floor-centrifuge, use sterile
13-ml Sarstedt round-bottom polypropylene tubes with push
caps (Sarstedt Inc., Newton, NC).
6. Chloroform (Sigma-Aldrich, St. Louis, MO). A separate,
dedicated 100- or 500-ml glass bottle with chloroform labeled
24 Elke M. Wagner

“RNA only” is strongly recommended. Avoid skin contact,

wear gloves, and, if possible, work in a hood. Use appropriate
thick-walled waste containers, as organic compounds like chlo-
roform dissolve some plastic containers (and plastic bags)
readily!
7. Any 5-ml glass pipettes, graduated (attention: do not dip too
far into the liquid, graduation marks dissolve in chloroform!).
8. Isopropanol (Sigma-Aldrich). A separate, dedicated 100- or
500-ml bottle of isopropanol, labeled “RNA only” is highly
recommended. Avoid skin contact with isopropanol, wear
gloves, and if possible, work in a hood.
9. Absolute ethanol (Sigma-Aldrich). A separate, dedicated 100-
or 500-ml bottle of absolute ethanol labeled with “RNA only”
is strongly recommended. Prepare 50 ml 75 % alcohol for
washing RNA, made up with RNase-free water, and store in a
50-ml Falcon tube.
10. Refrigerated centrifuge for centrifugation up to 12,000 × g,
e.g., Eppendorf 5417R (Fisher Scientific).
11. RNase-free tubes, 1.5 and 2 ml (e.g., Safe-Lock Eppendorf;
Fisher Scientific). Use with clean gloves. Dedicate a box or bag
of tubes for work with RNA only. Keep bag closed.
12. Thermal block for microcentrifuge tubes set to 55–60 °C.

2.2 Isolation of RNA 1. RNaseZap (Ambion Inc.).

from Cultured Cells 2. A clean (“RNaseZap”-wiped) tabletop centrifuge for micro-
and RNA Cleanup centrifuge tubes.
Using the Qiagen
3. Individually wrapped cell scrapers (e.g., BD Falcon, 18-cm
RNeasy Mini Kit handles), one per cell culture plate.
4. QIAshredder (Qiagen, Valencia, CA) for homogenization (one
per cell culture plate).
5. A few milliliter of b-mercaptoethanol (Sigma-Aldrich). This
reagent should be stored in the refrigerator. Use only in a
hood, or keep on ice if no hood is available. Work fast, close
containers quickly, and get rid of waste in a closed container/
bag.
6. RNeasy Mini Kit (Qiagen). Restore buffers with absolute etha-
nol as indicated in the kit.
7. Qiagen RLT buffer (included in the kit). Prepare the necessary
amount in a 15-ml tube: 350 ml per sample for tissue RNA
cleanup; 600 ml per sample for RNA extraction from cells. Add
10 ml of b-mercaptoethanol per 1 ml of RLT buffer under a
hood.
8. RNase-free DNase (Qiagen No. 79254): Add 550 ml of RNase-
free water to the lyophilized DNase I. Mix gently; do not vortex!
Aliquot in 5 × 110 ml portions (you will need 10 ml per sample)
 Monitoring Gene Expression: Quantitative Real-Time RT-PCR 25

and freeze at −20 °C. Thawed aliquots can be stored at 4 °C

for up to 6 weeks. Frozen aliquots can be stored at −20 °C for
9 months. Buffer RDD is stable in the refrigerator for at least
9 months.

2.3 Quantification 1. UV spectrophotometer (or equal instrumentation, for example

and Storage of Total a NanoDrop ND-1000 spectrophotometer, Thermo Scientific,
RNA Chicago, IL).
2. Clean quartz microcuvette(s) with 100 ml volume for UV
spectrophotometer.
3. Aliquots of RNase-free water (e.g., 100 ml, Ambion Inc.).
4. RNase-free 1.5-ml tubes for quantification.
5. Pressure air to dry cuvette between samples, if available.
6. Ultra Pure 1 M Tris–HCl, pH 7.5 (Invitrogen).
7. RNase-free 10 mM Tris–HCl, pH 7.5: to prepare, dilute 1 ml
Ultra Pure 1 M Tris–HCl, pH 7.5 with 99 ml of RNase-free
water.
8. Aerosol-tight pipette tips for pipetting RNA, cDNA, or other
RT-PCR components.
9. Optional: Prepare 1 or 1.5 % agarose gel(s) with RNase-free
buffer to check quality of RNA samples. Any 1–1.5 % agarose
gel made up with RNase-free gel reagents (agarose, water, buf-
fer) will do. Use 0.5× TAE as gel/running buffer, and ethidium
bromide or any dye used in your lab to visualize nucleic acids.

2.4 Reverse 1. TaqMan Reverse Transcription Reagents (Part no. N808-

Transcription (RT) 0234, protocol no. 402876, ABI). Just before use, prepare a
bucket with wet ice. Thaw the components of the mix, spin
down, and keep on wet ice. Keep the enzyme in the freezer
until ready to use. Use random primers, not oligo(dT) (see
Note 1). Read kit instructions before use; versions/concentra-
tions may change.
2. Aerosol-tight pipette tips for pipetting RNA, cDNA, or other
RT-PCR components.
3. Molecular biology grade water (Ambion Inc.).
4. Thermo cycler programmed as follows: 10 min at 25 °C,
60 min at 37 °C, 5 min at 95 °C.
5. 200-ml Thin-walled RNase-free tubes that fit the thermo cycler
(if necessary, use an adaptor plate).

2.5 Real-Time 1. MicroAmp 96-well optical reaction plate (ABI) and MicroAmp
PCR (qPCR) optical adhesive film (ABI; part number 4314320, MicroAmp
adhesive film applicator, part number 4333183). Touch only the
sides of the cover films! Impurities such as smudges absorb light
and will lead to incorrect readings and quantitations of the reac-
tions in the sample wells.
26 Elke M. Wagner

2. Dark plate support (ABI, 96-well plate support base, part

number 4379590).
3. TaqMan® Gene Expression Assays (for the choice of primers see
Notes 3–6). Keep frozen until use, thaw aliquots, and keep on
wet ice, and keep dark (e.g., cover tubes in aluminum foil).
A traditional assay for many lipoprotein researchers will be to
detect the murine low-density lipoprotein receptor (LDLr,
e.g., Mm00440169_m1, ABI) and b-actin as the endogenous
control (4352341E, ABI) in liver or macrophages.
4. TaqMan® Universal PCR Mastermix w/o UNG (see Note 7)
(Part no. 4324018, protocol no. 4304449, ABI) on wet ice.
Store in refrigerator after thawing.
5. 2 % Agarose gel to check specificity of assays/tissues used for
the first time. Use 0.5× TAE or 1× TBE as gel/running buf-
fer, use ethidium bromide or any dye used in your lab to visu-
alize nucleic acids, and use a 100-bp ladder as size standard
(e.g., Gene Ruler™ Ultra Low Range, Fermentas, Glen
Burnie, MD).

3 Methods1

RNA is readily degraded by RNase, an enzyme present ubiqui-

tously. Therefore, it is critical to work clean and fast, from the
beginning to the end of the procedure. When working with mice
(see Note 8), avoid bleeding of tissues as far as possible; use only
clean tools and wear a clean lab coat, gloves, and a mask. RNA
isolation always requires fast treatment, but RNA from tissues such
as lung, intestine, and adipose tissue are especially prone to degra-
dation. Tissue pieces more than 5 mm × 5 mm in size will not be
totally immersed with RNAlater, as the passive diffusion of the
liquid will not proceed that far. Therefore, prepare several small
pieces of tissue, immerse in RNAlater, and snap-freeze samples as
well. For adipose tissue, consider using Allprotect (Qiagen) in
place of RNAlater.
For RNA isolation from cells: A confluent 100-mm Petri dish
will hold a few million cells, depending on the size and confluency.
Lipid-loaded fat cells are quiescent and very large, and more than
one confluent 100-mm plate might be needed per experimental
condition. For liver cells or macrophages, one 60- or 100-mm dish
will provide enough RNA for qPCR. For example, pooled perito-
neal macrophages from five unboosted mice, plated onto one

1
The processes described in this chapter were developed during the author’s
fellowships at the University of Graz, Austria, and the NHLBI, NIH, Bethesda,
MD, USA. The processes described here do not represent procedures specified
by Baxter.
 Monitoring Gene Expression: Quantitative Real-Time RT-PCR 27

100-mm Petri dish in fresh medium and rinsed twice after 3 hours,
provide ~10 mg total RNA. Read the Qiagen RNeasy Handbook
carefully. It provides a valuable overview of the considerations
involved in RNA work, as well as the use of common cells/tissues
for RNA extraction (e.g., table of RNA quantities extracted from
common cell lines).
We assume that the user is familiar with basic methods in
molecular biology, such as running agarose gels and performing
enzymatic assays (RT-PCR).

3.1 Total RNA Isolation and stabilization of mouse tissue is described, but the
Extraction from Tissue methodology is applicable to any mammalian species.
Using TRIzol
1. Wipe work space, glass plate, scissors, and forceps with
3.1.1 Isolation and RNaseZap and 70 % ethanol before handling each mouse. Pre-
Stabilization of Tissue label RNase-free tubes containing RNAlater—for example,
1.5 ml RNAlater per 2-ml tissue-sample tubes. Tubes are kept
closed on clean tube racks. Optionally, larger tubes can be
used, and up to 10 ml RNAlater may be added to fully immerse
precious samples (a 10- to 15-fold volume of RNAlater is rec-
ommended by the manufacturer—still, pieces that are too large
will not be immersed by diffusion).
2. (Optional: Pin down dead mouse body on Styrofoam lid).
Wipe mouse abdomen with alcohol. Cut open outer belly
skin/fur by making an X-like incision on the belly towards the
legs. Flip skin/fur to the side (optional: and pin down).
Abdomen is now free.
3. Use a new pair of scissors and forceps to open the abdominal
wall. Excise a piece of tissue. Work from fat and intestine to
lung and liver (liver bleeds most).
4. Liver must be cut into small pieces up to 5 mm × 5 mm (one
piece per extraction is enough), intestine into 15-mm-long
pieces, and lung, fat, and brain into 3–4 pieces of 5 mm × 5 mm
each per sample. Intestine needs to be cleaned by carefully
striking out the diet with forceps onto a clean glass plate.
Excision of brain tissue is tricky and needs practice: have two
bent forceps to get the whole brain out immediately after the
skull is opened crosswise with sharp scissors. Work fast.
5. Always immerse tissue immediately in RNAlater solution and
throw cups into liquid nitrogen or shock-freeze in a dry ice/
ethanol bath (the latter dissolves ink labels on tubes—make
sure tubes are labeled on lid as well). Store at −70 °C till fur-
ther use.
6. Work through steps 1–5 above for each mouse, and then pro-
ceed to the next mouse.
7. Frozen tissue in RNAlater is stable for several months. Thus,
RNA extraction from these tissues can be done any time after
excision and storage of tissues.
28 Elke M. Wagner

3.1.2 RNA Extraction 1. For RNA extraction from tissue, prepare a clean workspace
from Tissue (TRIzol) where homogenization takes place.
2. Set up the Polytron/Ultra-Turrax, rinse the homogenizer with
RNaseZap or 70 % cleaning alcohol, and wipe dry before each
new sample.
3. Wearing clean gloves, pre-label the 12-ml tubes for
homogenization (see Note 9), add 1 ml TRIzol, and keep
tubes on ice. I recommend not isolating more than 8–12 sam-
ples at a time. If samples are still degraded, try isolating fewer
samples at a time, working faster and make sure samples are
kept clean and cold.
4. Wearing clean gloves, pre-label RNase-free 1.5-ml tubes for
storage of the isolated RNA samples. For number of tubes/
aliquots see Subheading 3.3, step 9.
5. Preheat a thermal block for microcentrifuge tubes to 55–60 °C.
6. Thaw tissues once everything is prepared for RNA extraction
(see Subheading 2). Add 50–100 mg tissue per 1 ml TRIzol
(not more than 10 % w/v per sample). Pick the pieces out of
the tube and weigh them. If necessary, chop off excess pieces
of tissue until the desired weight is reached.
7. Homogenize tissues in TRIzol with Polytron/Ultra-Turrax
for 5–10 s. Place back on ice immediately. After another 20 s of
chilling, homogenize for a second time. Rinse homogenizer in
RNase-free water (dip in 50-ml Falcon tube with water, turn
on briefly), and then rinse with 70 % alcohol. Wipe dry.
8. After all samples are homogenized in TRIzol, let samples sit at
room temperature for 5 min.
9. Add 0.2 ml chloroform, mix by vigorous shaking, and let stand
at room temperature for 2–3 min.
10. Centrifuge for 15 min at 4 °C at 12,000 × g (see Note 10).
11. Transfer colorless upper phase containing the RNA into a new,
labeled centrifugation tube.
12. Precipitate RNA with 0.5 ml isopropanol. Let stand for 10 min
at room temperature.
13. Centrifuge for 10 min at 4 °C at 12,000 × g.
14. The RNA pellet is a colorless, often invisible pellet on the bot-
tom of tube, against the outer wall of the tube. Carefully dis-
card supernatant, holding the tube with the pellet up so that
the risk of discarding the pellet is minimal. Carefully wash pel-
let with 1 ml of 75 % EtOH, vortex, and centrifuge at max
7,500 × g for 5 min at 4 °C.
15. Briefly air-dry RNA pellet (5–10 min; place tube upside down
on tissue, making sure pellet remains in the tube). Do not let
the pellet completely dry out.
 Monitoring Gene Expression: Quantitative Real-Time RT-PCR 29

16. Dissolve pellet in RNase-free water, resuspend by pipetting,

and transfer into 1.5 ml tube. For adipose tissue, intestine, and
lung use 100 ml RNase-free water (up to 300 ml may be used).
For liver, preferably 500 ml (but up to 1,000 ml) RNase-free
water may be used.
17. Incubate at 55–60 °C for 10 min; all of the pellet should have dis-
solved. Put back on ice. Adding more water, reheating and vortex-
ing may help to dissolve the pellet. However, if the sample doesn’t
dissolve (e.g., because it was too dry) it must be discarded.
18. Measure concentration at 260 and 280 nm according to
Subheading 3.3 and proceed to RNA cleanup.

3.2 Total RNA 1. Adjust the volume of about 5–20 mg RNA (depending on the
Extraction from Cells availability and volume of the isolated RNA) to 100 ml with
and RNA Cleanup RNase-free water (or use 100 ml of the original RNA extract, if
Using the Qiagen total RNA content <100 mg/100 ml).
RNeasy Mini Kit 2. Add 350 ml of buffer RLT (containing b-mercaptoethanol),
3.2.1 For Cleanup
and mix well.
of Total RNA from Tissue 3. Add 250 ml of absolute ethanol and mix well by pipetting. Do
After TRIzol Extraction not vortex. Proceed with step 6 below (application to spin
column).

3.2.2 Cell Culture, 1. Label RNase-free 1.5-ml microcentrifuge tubes, have neces-
Adherent Cells sary aliquots of RLT buffer with b-mercaptoethanol prepared,
and proceed to cell culture hood.
2. To harvest adherent cells, e.g., 3T3-L1 fat cells, HepG2 liver
cells, or murine peripheral/abdominal macrophages, remove
medium by aspiration and rinse cells to eliminate dead cells or
red blood cells (hemoglobin may inhibit enzymatic reactions).
3. For one confluent Petri dish (100 mm) add 600 ml of RLT buf-
fer containing b-mercaptoethanol, collect lysate with cell scraper,
and transfer into a pre-labeled RNase-free tube. Genomic DNA
makes the lysate viscous at this stage, so use a wide-bore pipette
tip for transfer. Proceed back to the lab bench.
4. Homogenize the lysate by transferring 600–700 ml onto a
Qiashredder and spin for 2 min at full speed at room tempera-
ture. Discard shredder column. If more homogenate is avail-
able, use a new Qiashredder and repeat step.
5. Add 600 ml of 70 % EtOH to the homogenate and mix by
pipetting. Do not vortex.
6. Apply 700-ml aliquots of the homogenate (or tissue RNA after
Trizol extraction from above) onto the pre-labeled spin col-
umn (RNA binding capacity is 100 mg), and spin at ³8,000 × g
for 15 s to bind RNA to membrane (see Note 10). Discard
flow-through (or use new collection tube—check with your
30 Elke M. Wagner

current Qiagen RNeasy Mini Kit Handbook Manual for the

amount of collection tubes provided). If more than 700 ml of
homogenate are present, repeat loading of the same spin
column.
7. Make sure all buffers are restored with the proper amount of
ethanol before first use. Mix by inverting. Add 350 ml of buffer
RW1, and spin for 15 s at ³8,000 × g.
8. DNase treatment on column: Prepare a master mix containing
70 ml of RDD buffer and 10 ml of DNase stock solution per
sample and mix gently. Do not vortex. Carefully add 80 ml of
the master mix onto the middle of the membrane without
touching membrane, and incubate at RT for 10–15 min. Do
not incubate more than 15 min.
9. Add 350 ml of buffer RW1, spin for 15 s at ³8,000 × g, and
discard collection tube.
10. Wash with 500 ml of buffer RPE, spin for 15 s at ³8,000 × g,
discard flow-through, or use new collection tube. Add again
500 ml of buffer RPE, spin for 2 min at ³8,000 × g, and discard
flow-through again. Spin another minute at full speed to air-
dry membrane.
11. Pre-label elution tubes (provided with kit). Discard collection
tubes and place the spin column on a new, pre-labeled, 1.5-ml
tube RNA elution tube.
12. Elute: Carefully add 30 ml of RNase-free water onto the middle
of the membrane without touching it, incubate for 1 min, and
spin for 1 min at ³8,000 × g (see Note 11). Repeat this step.
Discard the spin column and keep the tubes with the eluted
RNA on wet ice.

3.3 Quantification Eluted total RNA in RNase-free water can be quantified as such on
and Storage of Total a spectrophotometer or any instrument capable of UV spectros-
RNA copy. It is important to dilute the RNA in a neutral solution, pref-
erably a buffer, or water. Wear gloves. The purity of the RNA is
measured through the ratio of the absorption at 260–280 nm
(A260/A280). This value should come close to 1.8 (optimally 2) to
assure clean RNA. However, traces of protein impurity (e.g.,
DNase) will reduce this value (see Note 12). If necessary, dilute
samples several times, such that the absorption at 260 nm falls
between 0.3 and 1. Note that dilution factors may vary between
tissues.
1. Label RNase-free tubes and add 98.0 ml of 10 mM Tris–HCl,
pH 7.5 (or RNase-free water).
2. Add 2.0 ml of RNA, and mix. Put RNA tube back on ice.
3. Vortex and spin down.
 Monitoring Gene Expression: Quantitative Real-Time RT-PCR 31

4. Choose an RNA quantification program on your UV

spectrophotometer, or measure each sample first at 260 nm,
and then at 280 nm.
5. Blank the UV spectrophotometer with 100 ml of the Tris buf-
fer (or the RNase-free water) in a 1 cc (1 ml) quartz cuvette.
6. Measure the absorbance of the samples, rinse in between with
RNase-free water, and blow-dry with pressured air, if available. If
necessary, repeat samples in different dilutions if absorbance at
260 nm (A260) is out of range (less than 0.3 or greater than 1).
7. RNA concentration (mg/ml) = 44 mg/ml × dilution factor ×
absorption at 260 nm (A260), where 44 mg/ml is the concen-
tration for RNA with an A260 = 1 at neutral pH in a 1-cm
pathway cuvette. When diluting 2.0 ml into 100 ml of total
volume, the dilution factor is 50.
8. Make sure the purity is acceptable (A260/A280 = ~1.6–2.0).
9. Store the purified RNA at −80 °C in aliquots of 2–6 mg for
precious RNA, or in larger portions for abundant RNA sam-
ples (see Note 13 for the amounts of RNA that will be
needed).
10. RNA integrity is best checked by agarose gel electrophoresis.
Use ~2–3 mg of RNA. One strong band for the 28S rRNA
and one band for the 18S rRNA that is about half as intense
as the 28S rRNA band, as shown in Fig. 2, shall be present
(see Note 14). The 5S rRNA band is usually weak.

3.4 Reverse Total RNA is reverse-transcribed according to the user manual

Transcription (RT) (TaqMan® Reverse Transcription Reagents, ABI). Two micrograms
of RNA are reverse-transcribed in a total volume of 100 ml [62.5 ml
master mix, 37.5 ml sample (2 mg RNA adjusted to 37.5 ml with
RNase-free water)], using random hexamers as primers. Wear
gloves.
1. Program thermo cycler.
2. Label tubes. Include one tube for the RT− control (no reverse
transcriptase included, see Note 14) and one extra tube for a
second reverse transcription of one of the normal/wild-type
samples used for the standard curve (see Note 15).
3. Thaw reagents and place on ice, except for the reverse tran-
scriptase enzyme, which should be stored in the freezer
(−20 °C) until use.
4. Calculate the volume of 2.0 mg RNA for each of your isolated
RNA samples to be reverse-transcribed. Subtract this amount
from 37.5 ml; this is the amount of RNase-free water you have
to pipette into the corresponding tube for reverse transcription.
(RNA will be added last.)
32 Elke M. Wagner

Fig. 2 Adipose tissue RNA after DNase cleanup. Approximately 3 mg of freshly

isolated RNA samples were adjusted to 20 ml with RNase-free water and were
subjected to agarose gel electrophoresis on a 1.2 % E-gel (Invitrogen)

5. Thaw RNA samples on ice.

6. Mix a master mix for all samples, plus one extra volume:
Per 1× reaction volume (100 ml), combine:
10 ml 10× buffer.
22 ml MgCl2.
20 ml dNTPs.
5 ml random hexamers.
2 ml RNase inhibitor.
Mix by vortexing.
Per 1× reaction volume, add 3.5 ml reverse transcriptase.
Mix carefully by pipetting. Avoid air bubbles or stressing the
enzyme by mechanical forces (such as vortexing).
7. Pipette 62.5 ml of Master Mix to each sample tube already con-
taining water. Add the 2.0 mg of the corresponding sample
RNA into the mix. This should result in 100 ml/tube.
8. In the RT− tube prepare one reaction volume without RT:
10 ml 10× buffer.
22 ml MgCl2.
20 ml dNTPs.
5 ml random hexamers.
2 ml RNase inhibitor.
3.5 ml RNase-free water.
Add 2 mg of the desired RNA for the RT− control in a volume
of 37.5 ml. Vortex.
 Monitoring Gene Expression: Quantitative Real-Time RT-PCR 33

9. Spin down tubes or flick tubes to ensure that no air bubbles

accumulate at the bottom of the tube (they inhibit heat trans-
fer). Enter the reaction volume and then start the reaction on
the thermo cycler.
10. Freeze RNA aliquots at −70 °C.
11. When the RT is finished, freeze cDNA at −20 °C, or use imme-
diately for real-time PCR (keep on wet ice).

3.5 Real-Time The use of the absolute quantification software module in order to
PCR (qPCR) do a relative quantification with standard curve is described (see
Note 16). The term “standard curve” may be misleading, if one is
thinking of an external standard in absolute numbers (e.g., exact
copy numbers). Here, one of the samples, typically a wild-type/
control sample, is used in a serial dilution to relate the RNA con-
centration (mg/ml) measured by UV spectroscopy with the Ct.
However, we will not know the absolute copy number of a tran-
script (now cDNA) present in our sample.
This protocol describes a single PCR setup (see Note 17) with
the detection of a housekeeping gene/endogenous control (see
Note 5) for normalization to the “RNA load” in separate reac-
tions. We also assume that the SDS7300 (ABI) with the corre-
sponding 7300 SDS version 1.3 software is being used and that the
user has access to Microsoft Office Excel. Any other qPCR machine
and software can be used. The sample description and assay setup
for a 96-well plate remain the same. An “assay setup run” to define
the dilution range for the new assay/tissue combination is per-
formed before running all samples (see Note 18). Fewer standards
are necessary for known assay/tissue combinations with preestab-
lished dilution ranges and thus the assay setup differs slightly (see
Note 19). When handling fluorescent assays, keep in mind that
energy excites fluorescence. Keep tubes dark (see Note 6) and cool.
For the choice of your primers and TaqMan assay, see Notes 3–5.

3.5.1 Program Setup 1. Start the SDS7300 and under file/new choose “absolute
quantification.” If software was already open, restart it.
2. Choose the appropriate assay (called “detector” when using
the ABI software). Make sure to choose the according reporter
dye (FAM, VIC, etc.) used in your assay. You may want to
define your assays first (new detectors).
3. Choose ROX as passive reference (included in buffer to nor-
malize for pipetting errors).
4. Put in sample setup for the “assay setup run” (see Note 18):
The SDS software analyzes data row-wise so it is easier to
design the sample application row-wise, from A1–A12,
B1–B12, etc. Each sample must be measured at least in dupli-
cate, preferably in triplicate. For example, when testing the
34 Elke M. Wagner

setup for murine LDL-receptor primers (ldlr) and b-actin as

housekeeping primers in a new tissue (see Note 18), a typical
plate layout might be:
Wells that will contain the ldlr Master Mix
– A1–B3 standard concentrations 1–5, each in triplicates.
– B4 RT− control.
– B5 NTC (non-template control = water).
Wells that will contain the b-actin Master Mix
– C1–D3 standard concentrations 1–5, each in triplicates.
– D4 RT− control.
– D5 NTC.
– (For an assay that has already been established, use the
layout described in step 10, below.)
5. Choose assay (detector) per well/rows: select wells, go to
view/well inspector, and check according assay (e.g., ldlr).
Repeat step for each assay.
6. Define sample status for each sample/row/selected wells in
the well inspector (unknown, standard, NTC). It is OK to
leave wells empty and still measure them (define as unknown
or NTC and assign an assay). Defining all wells of the plate
assures that samples are measured even if plate is mounted in
wrong position, e.g., well H12 in position A1; in this case,
detectors and sample status can be corrected later.
7. Enter the quantities for the standards in the well inspector
(e.g., for A1–A3 enter “100” for the theoretical 100 ng mRNA
per well, for A4–A6 enter “10” (see Note 18)).
8. Thermal program setting: On the Instrument Tab, check pro-
gram data and correct if necessary [1 × 10 min 95 °C; 40× (15 s
95 °C, 1 min 60 °C)]; add a 1 × 10 °C forever step (sample will
be used later for gel analysis when doing an assay setup for a
new assay/tissue combination).
9. Save setup; Choose a self-explaining name, e.g., date, user,
samples, and assay.
10. For setup of a preestablished assay/tissue combination, follow
steps 1–3. The plate layout differs from that in step 4 as fewer
standards are necessary. A typical plate layout for a preestab-
lished assay is outlined below, assuming that you test standards
and samples each in triplicates (see Note 19).
Wells that will contain the ldlr Master Mix
– A1–A9 standard concentrations 1–3, each in triplicates.
– A10–C3 samples 1–6, each in triplicates.
 Monitoring Gene Expression: Quantitative Real-Time RT-PCR 35

– C4 RT−.
– C5 NTC.
Wells that will contain the b-actin Master Mix
– D1–D9 standard concentrations 1–3, each in triplicates.
– D10–F3 add samples 1–6, each in triplicates.
– F4 RT−.
– F5 NTC.
– Follow steps 5–9, above, and then proceed to step 11,
below.

3.5.2 cDNA Dilution 11. Dilute cDNA as needed for the standards and the samples (see
Notes 18 and 19). Use siliconized 0.5 ml tubes. If dilutions
are to be stored, thaw them only once more (see Note 20).
12. Use a total PCR reaction volume of at least 25 ml per well on
the 96-well plate (see Note 21). A Master Mix with the
TaqMan® Universal PCR Master Mix w/o UNG is prepared
per assay (“detector,” e.g., ldlr primers).
For each 1× 25 ml reaction volume mix:
12.5 ml 2× buffer.
6.25 ml molecular biology grade water.
1.25 ml 20× ABI TaqMan® Gene Expression Assays (e.g.,
ldlr or b-actin).
Include one extra volume per every pipetted row (12 wells),
plus extra volumes for the NTC and the RT− controls.
For example:
– For an “assay setup run” (five standard concentrations in
triplicate = 15 samples, plus RT- and NTC controls) it is
necessary to mix 20× volumes of each master mix for each
assay (detector) master mix.
– For an established assay of 12 experimental samples (six
wild-type liver cDNA, six transgenic liver cDNA) and 3
standard curve dilutions, each in triplicates, calculate as
follows: 12 × 3 samples = 36, 3 × 3 standard curve = 9, plus 1
RT− and 1 NTC = 47. Add 4 extra volumes as you will
pipette ~4 rows on the plate = 51× volumes for each assay
(detector) Master Mix.
13. Place a labeled (barcoded) 96-well reaction plate onto the dark
support. Add 20 ml of the appropriate Master Mix into the
appropriate wells (on the side and close to but not touching
the bottom of each well).
14. Add 5.0 ml of the according sample into the mix. Cover with
optical film according to instructions, spin plate down briefly,
or flick samples down.
15. Disconnect computer from network when using ABI.
36 Elke M. Wagner

16. On the Instrument Tab change volume to 25 ml and hit

ENTER.
17. Save settings again.
18. Start PCR.
19. Freeze original cDNA samples at −20 °C after use.
20. After the qPCR is finished, you may immediately analyze your
data or you may want to transfer the data to another PC where
the software is installed (reestablish network if needed). If you
want to run another qPCR, immediately restart the SDS
software before beginning another qPCR run (only for ABI
software). Cool/freeze the finished plate in the dark for analy-
sis of bands on an agarose gel if not done immediately (see
Note 22). You should check the gel before running precious
samples with a new assay.

3.5.3 Analysis The SDS software analyzes data according to the settings defined
by the user. This is the advantage when doing the analysis “manu-
ally.” You will define the settings, meaning the range of “baseline”
(background fluorescence), and the wells to be analyzed together
(usually you analyze per detector, as the PCR efficiency and linear-
ity may differ between assays). Once the software has calculated all
Cts, we will export the raw data to, e.g., Excel, and proceed there.
21. In the data file, go to Results, amplification plot. Select the
samples per assay/detector (e.g., choose all wells with the ldlr).
Omit wells of assays that are not presently analyzed (select
wells, in View/well inspector, and check omit).
22. Choose manual baseline. Set the baseline using the linear view
of the results/amplification plot—change from “Delta Rn vs.
Cycle” to “Rn vs. Cycle” on the upper right side of the window
(see Fig. 1b). One can see the end of the baseline/background,
where the fluorescence starts rising. Common settings for
the baseline are between Cts 3 and 15, e.g., 3–12, leaving
enough space before fluorescence comes up. Leave at least
three Cts between end of baseline and start of exponential
phase, to be seen in the “linear” view Rn vs. Cycle; see Fig. 1b.
Otherwise, an undesirable “drop” in the baseline/background
can occur; see Fig. 3. The minimum baseline setting is Cts 3–6.
Hit enter.
23. Go back to the “Delta Rn vs. Cycle” view. Set the threshold:
move the threshold line to the middle of the “linearized” PCR
range where all reactions of this assay seem to run parallel.
24. Hitting the analysis button, threshold line becomes green.
25. The intersection of the threshold line and the sample curve is
called cycle at threshold (Ct); see Fig. 1b. It is an inverse
measure of the mRNA concentration, with less cycles (lower Ct)
 Monitoring Gene Expression: Quantitative Real-Time RT-PCR 37

Fig. 3 Upper panel : Correct manual baseline setting. Lower panel : Incorrect baseline setting. Background was
defined too close to exponential phase

corresponding to more mRNA transcripts present in the

original sample.
26. Check the standard curve tab: a value of −3.33 for the slope
indicates that between one log of sample concentration (ten-
fold difference) are 3.33 cycles (or Cts), which corresponds to
a doubling of DNA per cycle (optimal reaction efficiency,
38 Elke M. Wagner

23.33 = 10). Efficiencies are important for the DDCt method; see
Note 16. The R2 value is a measurement of the linearity, and
should be close to 0.99.
27. All values appearing in the report tab represent quantities at
that according threshold (reporter dye intensity).
28. In the report tab, export data. All samples that are not omitted
when hitting the analysis button will be exported into a comma
delimited .csv file. In Excel, the .csv file can be opened (choose
“all files” in the OPEN window under “files of type:”) and
converted by hitting “next.” When doing this the first time,
check for appropriate settings, comparing the original data
with the Excel data. Save file as an Excel worksheet. Repeat the
analysis for each assay/detector.
29. At the end, copy all relevant sample data such as run
specifications, sample names, well positions, quantities, and Cts
in one Excel master spreadsheet (see Note 23). Although Cts
won’t be “used” in this protocol, they represent a control for
the quality of the PCR. If you use sample data with a Ct between
35 and 40, you should be critical and decide if you want to use
this value—are the curves representative, do the triplicates
agree, is the Ct within the range of the standard curve?

3.5.4 Calculation In our example we were interested whether the expression of the
of Relative Gene LDLr in a transgenic liver was upregulated or downregulated com-
Expression Levels pared to the wild-type liver (wild-type is defined as 1, or 100 %
expression), see also Note 24:
30. Divide the quantities of each sample by the quantity of the
according sample’s housekeeping gene (e.g., qty ldlr sample1/
qty b-actin sample1). The column containing these data can be
named “normalized quantities.”
31. Let’s stick with the experiment of six wild-type and six trans-
genic mice: calculate the mean + standard deviation of the
“normalized quantities” of each group. (Exclude assays that
haven’t worked properly.)
32. Divide both numbers by the mean of the wild-type samples. The
mean of the wild-type samples will now become 1, as it was
divided by itself. The mean of the transgenic livers will now
have an x-fold higher or lower expression level compared to
the control group (e.g., 0.2-fold means that 20 % of the wild-
type level are present, meaning 80 % downregulation).
33. The standard deviations of the mean quantities must be divided
by the mean of the control group as well, in order to give the
appropriate standard deviations (Note 25).
MIQE guidelines [15] should be followed when publishing
results of quantitative real-time PCR experiments.
 Monitoring Gene Expression: Quantitative Real-Time RT-PCR 39

4 Notes
1. Random hexamers are used as they guarantee equal reverse
transcription of all mRNAs, while polyA can be used as primer
only if the sequence to be amplified later is close to the 3¢ end.
However, since one is never sure which gene sequences have to
be measured using the same cDNA further down the line, we
recommend random hexamers.
2. ABI, as well as other companies, has designed primer and probe
sets for virtually all human and mouse genes, and custom prim-
ers can be produced as well. The predesigned and specific
“TaqMan® Gene Expression assays” are popular and are an
excellent choice for the analysis of gene expression by RT-qPCR.
See also Note 4 for more details.
3. The choice of the primers/probe (also called qPCR primers,
primer set) for a TaqMan® assay must be carefully chosen. In
general, a region of 60–100 nts on the gene of interest (mRNA)
is chosen, where two traditional PCR primers and one probe
that binds in between are designed. If designing your own
assay, get acquainted with the primer-design software (e.g.,
Primer Express, ABI). Exon–exon spanning probes are pre-
ferred for assays (see Note 4), as they exclude amplification of
introns, e.g., from genomic DNA impurities. A gene with
many isoforms or transcript sizes needs thorough examination
of the sequences regulated and expressed in the relating tissue.
The number of transcripts which a specific assay detects can be
looked up for pre-developed assays from ABI—it is recom-
mended to use an assay that detects the ref-sequence or the
most transcripts known at present. In some cases, for example,
ABCG1 which has many tissue-specific transcripts, it may be
wise to use more than one assay, if real-time PCR is the tech-
nique of choice for mRNA quantification.
4. If you go to the ABI webpage and browse to the product
guide, you can choose to search for gene expression assays
(pre-developed) [16]. Enter only systematic names (low-density
lipoprotein receptor), not common short names (LDLr). The
extension “_m1” in ABI pre-developed gene expression assays
refers to an exon–exon border-spanning assay, while
“_g1” refers to detection of an exon–exon border which
includes a short intron. The extension “_s1” refers to detec-
tion of genomic DNA. In the last two cases, DNase treatment
of RNA is obligatory in order to not also detect genomic DNA.
If you run your qPCR samples on a gel after using a new primer
set/assay for the first time, and more than one PCR product is
visible on the agarose gel, you need to increase the DNase
treatment or use another assay, or make sure that there are no
40 Elke M. Wagner

other transcript sizes (do a Northern blot analysis). An assay

will most reliably exclude amplification of introns when chosen
towards the 5¢ end of a sequence (long introns). If ordering
assays from ABI, become acquainted with the ABI home page,
call the helpline and have the search options explained well.
5. The actual RNA (or cDNA) “loaded” per well is determined
by measuring an endogenous control or “housekeeping gene,”
which is constitutively expressed in a tissue. However, endog-
enous controls can have different properties in different tis-
sues, so the endogenous control chosen for a given experiment
must be appropriate. Housekeeping genes/endogenous con-
trols for liver, macrophages, intestine, lung, adipose, and brain
are as follows: (1) b-actin—note, however, that b-actin is nei-
ther recommended in adipose tissue nor in macrophages which
have not been equally differentiated in vitro, due to different
cell sizes/actin levels; (2) HPRT1—it is more highly expressed
in brain, but is constitutively expressed in other tissues; and
(3) 18S rRNA for the detection of very abundant genes of
interest. The difference in cycles between the endogenous
control and the gene of interest should not exceed 12 cycles in
a sample; keep this in mind when measuring low-abundance
genes, e.g., in liver, where 18S rRNA is very abundant. GAPDH
is not a good choice, as it is involved in carbohydrate metabo-
lism, which is linked to fat metabolism. Neither is cyclophilin,
which may interact with the immune system. For testing of
various endogenous controls, check available pre-developed
assay sets (e.g., TaqMan® Express Endogenous Control Plate,
for human samples, ABI [17]).
6. The TaqMan® Gene Expression Assays come as a 20× primer/
probe mix that will give a final PCR concentration of 900 nM
primer and 250 nM FAM-labeled probe. Aliquot into six por-
tions after the first thawing. Use dark-colored tubes, optionally
wrap in aluminum foil while used. Aliquots can be used for
several (at least 3) years when properly stored at −20 °C, and
can be thawed up to ten times.
7. UNG is an enzyme that digests specifically amplicons contain-
ing dUTP instead of dTTP. If you use dUTP for your qPCR,
you can control for contamination of end products when using
UNG in your PCR buffer. Keep in mind that negative controls
(NTC, water) are still a must even when using UNG.
8. Mice are active at night; therefore, a metabolically active liver
might be obtained at night. Consider whether it is necessary to
fast mice before collecting tissue.
9. Brain tends to foam extremely. It is wise to use at least 10- to
12-ml tubes.
 Monitoring Gene Expression: Quantitative Real-Time RT-PCR 41

10. Conversion of ×g (times relative centrifugal force; gravity) to

rpm (revolutions per minute): rpm = sqrt [(1.118 × 105 × g)/r],
where r is the radius of the rotor used (in cm).
11. For centrifugation of the tubes, place elution tube with spin
column in centrifuge, leave two positions spare, place next elu-
tion tube into position, and so forth. Turn tube lids to the side
over the free positions, in order to support them during cen-
trifugation. Clean rotor lid inside with 70 % ethanol; let dry.
Close rotor lid, and spin.
12. For qPCR it will be acceptable if the ratio of absorption at
260–280 nm is lower, or if RNA is partially degraded (slight
smear between 28S and 18S). The parallel measurement of the
gene of interest and an endogenous control/housekeeping
gene will control for RNA load during the qPCR. In general,
after a few attempts at isolating RNA, handling becomes better
and RNA cleaner (see Fig. 2). Fatty tissues like lung, fat, and
intestine will easily be degraded when more than one tissue is
taken from one animal at a time.
13. Two micrograms of RNA are used per reverse transcription
reaction. Thawing more than three times is not recom-
mended; discard RNA thereafter. RNA will be stable for at
least 12 months at −80 °C under these conditions. For dilu-
tions or small amounts (e.g., <1 mg/ml) of RNA or cDNA
use 0.5 ml siliconized/low retention microcentrifuge tubes
(Fisher Scientific) for storage. Do not thaw diluted samples
more than once.
14. Contamination of RNA with genomic DNA can sometimes be
seen on the agarose gel as a large band close to the origin. For
a qPCR assay using probes that do not span exon–exon bor-
ders over large introns, DNase treatment is an essential step
and must be carried out carefully. Furthermore, the use of a
“minus reverse transcriptase” (RT−) control reveals amplification
of genomic DNA. (Note also that an unusually low efficiency
of amplification in a sample containing all components, includ-
ing RT and cDNA, might indicate unspecific competition by
large quantities of genomic DNA.) Each set of RNA isolations
should have an RT− control included when doing the RT
step.
15. For the standard curve, one defined cDNA sample will be
used as a standard that will require larger quantities. It is rec-
ommended to add one extra RT reaction in order to reverse-
transcribe this “normal” sample (e.g., a wild-type sample)
twice and make sure one has enough material to work with.
16. Although we do a “relative” quantification of two or more con-
ditions, we use the ABI “absolute” quantification module. This
is preferred to the relative quantification module, as it is a less
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 The Project Gutenberg eBook of Chambers's
Journal of Popular Literature, Science, and Art,
Index for 1884
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Title: Chambers's Journal of Popular Literature, Science, and Art,

Index for 1884

Author: Various

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*** START OF THE PROJECT GUTENBERG EBOOK CHAMBERS'S

JOURNAL OF POPULAR LITERATURE, SCIENCE, AND ART, INDEX
FOR 1884 ***
[Transcriber's Note: This book contains links to individual issues of
Chambers's Journal contained in the Project Gutenberg collection.
Although we verify the correctness of these links at the time of
posting, these links may not work, for various reasons, for various
people, at various times.]
 CHAMBERS’S JOURNAL

OF

POPULAR LITERATURE

Science and Arts

1884

W. & R. CHAMBERS
LONDON & EDINBURGH

Edinburgh:
Printed by W. & R. Chambers.

[Transcriber's Note: This cross reference of issue number to page

number was provided by the transcriber.
Issue Pages Date
No. 1 1-16 January 5, 1884
No. 2 17-32 January 12, 1884
No. 3 33-48 January 19, 1884
No. 4 49-64 January 26, 1884
No. 5 65-80 February 2, 1884
No. 6 81-96 February 9, 1884
No. 7 97-112 February 16, 1884
No. 8 113-128 February 23, 1884
No. 9 129-144 March 1, 1884
No. 10 145-160 March 8, 1884
No. 11 161-176 March 15, 1884
No. 12 177-192 March 22, 1884
No. 13 193-208 March 29, 1884
No. 14 209-224 April 5, 1884
No. 15 225-240 April 12, 1884
No. 16 241-256 April 19, 1884
No. 17 257-272 April 26, 1884
No. 18 273-288 May 3, 1884
No. 19 289-304 May 10, 1884
No. 20 305-320 May 17, 1884
No. 21 321-336 May 24, 1884
No. 22 337-352 May 31, 1884
No. 23 353-368 June 7, 1884
No. 24 369-384 June 14, 1884
No. 25 385-400 June 21, 1884
No. 26 401-416 June 28, 1884
No. 27 417-432 July 5, 1884
No. 28 433-448 July 12, 1884
No. 29 449-464 July 19, 1884
No. 30 465-480 July 26, 1884
No. 31 481-496 August 2, 1884
No. 32 497-512 August 9, 1884
No. 33 513-528 August 16, 1884
No. 34 529-544 August 23, 1884
No. 35 545-560 August 30, 1884
No. 36 561-576 September 6, 1884
No. 37 577-592 September 13, 1884
No. 38 593-608 September 20, 1884
No. 39 609-624 September 27, 1884
No. 40 625-640 October 4, 1884
No. 41 641-656 October 11, 1884
No. 42 657-672 October 18, 1884
No. 43 673-688 October 25, 1884
No. 44 689-704 November 1, 1884
No. 45 705-720 November 8, 1884
No. 46 721-736 November 15, 1884
No. 47 737-752 November 22, 1884
No. 48 753-768 November 29, 1884
No. 49 769-784 December 6, 1884
No. 50 785-800 December 13, 1884
No. 51 801-816 December 20, 1884
No. 52 817-832 December 27, 1884
End of Transcriber Note.]

INDEX.
 Familiar Sketches and Essays.
Page
Acorns, Under the, 657
Brompton Cemetery, In, 753
Circulating-library Critics, 81
Conversation, the Art of, 442
‘Corners,’ 289
Furniture Saleroom, In a, 379
Girls, Wives, and Mothers, 33
Good-natured People, Mischief done by, 111
‘Grand Day,’ 561
‘Happy Ever After,’ 161
Heroines, 492
Highland Glen, In a, 511
Home-nursing, 417, 549, 609, 725
King of Acres, a, 12, 29
‘Kitchen Kaffir,’ the, 117
Literary Beginners, Another Word to, 49
Love, Concerning, 156, 333
Moor and Loch, On, 433
Mortality, Some Cheering Aspects of, 449
My Old College Rooms, 262
Nature on the Roof, 385
Newsmonger, the, 353
Norman Seascape, a, 390
River Holiday, a, 545
Saleroom, In a Furniture, 379
Spring Birds, 129
Væ Victis! 629
 Poetry.
Among the Daisies, 208
Angel Visitors, 224
Blackbird’s Nest, a, 448
Butterfly in the City, a, 368
Churchyard by the Sea, 128
Dawn of Peace, the, 592
Day in Early Summer, a, 704
Donald—a Pony, 144
Echoes, 256
En Passant, 560
Evening on the Lake, 480
Fairyland in Midsummer, 816
Hawthorn Story, a, 736
Hope On, Hope Ever, 768
July, 464
Last ‘Good-night,’ a, 496
Long Ago, 832
Love Lights, 160
Love-thought, a, 720
Michaelmas, 608
Mistletoe, 784
Modern Madrigal, a, 656
My Home in Annandale Revisited, 96
Night, 64
No Tears, 688
‘Not Beautiful!’ 176
‘Not Lost, but Gone Before,’ 48
Old, Old Story, 192
On the Coast, 544
One by One, 640
‘Only Cousins, don’t you see?’ 352
Parted, 400
Parting Words, 800
Quits! 320
Rhine Woods, In the, 384
Rime of Sir Lionne, the, 512
Serenade, 288
Six Little Words, 112
Solitary Singer, the, 304

Sonnets—
Love’s Watch, 16
Love’s Transfiguration, 16

Sonnets of Praise—
i. The Vales, 240
ii. The Mountains, 240

Spring in the Alley, 336

Story that Never Grows Old, a, 672
Stray Blossom, the, 576
To a Child, 80
Trifles, 432
’Twixt Daybreak and Daylight, 528
Water-ousel’s Song, 271
Wild-flowers from Alloway and Doon, 416
Wounder and Healer, 752
Year’s Wooing, a, 32
‘Yes,’ 624
 Popular Science.
Arsenic in Domestic Fabrics, 799
Colds, Common, 175
Colour-sense, 44
Electric Light, Curiosities of the, 140
Electricity and Gas, the Future of, 625
—— for Nothing! 453
Explosion, Story of a Vast, 705
Fuel, a New, 671
Gas Cooking-stoves, 367
Glacier Garden, a, 785
Marine Station, a Scottish, 465
Medicine, Common Errors in Domestic, 299
Microphone, Curiosities of the, 373
Month, The: Science and Arts—59, 124, 201, 265, 348, 412, 476,
557, 620, 685, 761, 825
Noxious Manufactures, 239

Our Health—
i. Health and its General Conditions, 113

ii. Food and Health, 234

iii. Some Food-dangers, and how to avoid them, 401
Parasitic Worms—Queer Lodgers, 278
Poisoning, 769
Science and Art School—Gordon’s College, Aberdeen, 183
Smoking Injurious to Health? Is, 78
Steel, 575
Steno-telegraph, the, 607
Water, 497
Weather, How it is Made and Forecast, 689
White-lead Manufacture, a New Process of, 158
 Tales and Other Narratives.
Abe, Story of, 817
Break-neck Venture, a, 588
Bushranger, Interviewed by a, 650

By Mead and Stream, a Story.—By Charles Gibbon, Author of Robin

Gray, Queen of the Meadow, The Golden Shaft, &c.
chap.

1. The Overture: ‘Much Virtue in If,’ 1

2. What might be, 2
3. What is to be, 20
4. In the Oak Parlour, 35
5. A New Eden, 37
6. Alone, 50
7. An Unloved Life, 67
8. ‘Will you speak that Word?’ 69
9. Slander’s Shaft, 82
10. Light and Shadow, 84
11. ‘Strictly Confidential,’ 99
12. A Fair Arbiter, 115
13. The Cares of State, 116
14. In Harvest-time, 132
15. The Banquet waits, 133
16. Light Hearts and Sad, 148
17. A Turning-point, 163
18. The Sealed Letter, 179
19. The First Interview, 181
20. Paved with Gold, 195
21. Dreams, 211
22. Home Again, 212
23. Changes, 227
24. The Work, 243
25. A Word in Season, 259
26. A Question of Division, 275
27. Why is she so? 291
28. ‘The Little Rift,’ 309
29. Suspicion, 323
30. Curious, 339
31. The Conjurer, 355
32. The Enthusiast, 356
33. Her Problem, 371
34. Judge Me, 388
35. The Maid was in the Garden, 403
36. Is it Too Late? 419
37. Down by the River, 436
38. Whirlwinds, 450
39. The other Side, 468
40. Madge’s Mission, 484
41. Pulled Up, 499
42. A Land Shipwreck, 517
43. Other People’s Money, 531
 44. An Apple of Discord, 547
45. High Pressure, 564
46. Downhill, 580
47. Under-currents, 595
48. Anxieties, 612
49. At Midnight, 614
50. A Crow to Pluck, 627
51. Hey, Presto! 642
52. How it was Done, 659
53. Pansy, 675
54. Poor Comfort, 692
55. Sweet are the Uses of Adversity, 707
56. Uphill, 723
57. The Secret in the Oak Parlour, 740
58. Clearing Up, 754
59. Glimpses, 756

Chewton-Abbot.—By Hugh Conway, 280, 295, 315

Colonel Redgrave’s Legacy, 780, 793, 811
Frendraught, the Fire of, 52
Gentleman of the Road, a, 429
Greenroom Romance, a, 471
Haunted Bridge, the, 814
In a Flash, 520
Last of the Stuarts, the, 600, 617
Miner’s Partner, the, 138, 152, 168, 185
Miss Marrable’s Elopement, 188, 198
Missing Clue, The, a Tale of the Fens—
chap.

1. The Arrival at the Saxonford Arms, 701

2. The Jacobite, 702
3. The Events of a Night, 716
4. After Fifteen Years, 718
5. The Colonel’s Daughter, 732
6. Hobb Dipping Bewildered, 749
7. Reginald’s Story, 751
8. The Search, 764
Mr Pudster’s Return, 569, 584
My Fellow-passenger, 252, 263
Nameless Romance, a, 541

One Woman’s History, a Novelette.—By T. W. Speight— 631, 646,

664, 680, 696, 710, 728, 743, 757, 771, 786, 804, 819
Queen Margerie, 677
Queer Company, In, 444, 461
Run for Life, a, 488
Silas Monk: a Tale of London Old City, 361, 374, 394, 407
Sketch from my Study Window, 343
‘So Unreasonable of Step-mother!’ 45
Terribly Fulfilled, 424, 440, 454

Two Days in a Lifetime.—By T. W. Speight— 5, 25, 41, 54, 73, 87,

105, 119
Vermudyn’s Fate, 536, 552
Witness for the Defence, a, 217, 231, 247
Yarn of the P. and O., a, 503
Zulu Romance, a, 330
 Notices of Books.
King Country, or Explorations in New Zealand, by Mr J. H. Kerry-
Nicholls, 637
Mushroom-growing, by Mr Wright, 501
Nature near London, by Mr Richard Jefferies, 225
Norfolk Broads and Rivers, by Mr G. Christopher Davies, 273

Book Gossip—
Aileen Aroon, by Dr Gordon Stables, 63
Anglo-Saxon Literature, by Mr John Earle, Rawlinson Professor
of Anglo-Saxon, Oxford, 411
Arminius Vambery, his Life and Adventures, 207
Athole Collection of Dance Music of Scotland, by Mr James
Stewart Robertson (Edradynate), 208
Chapter of Science; or, What is the Law of Nature? by Mr J.
Stuart, Professor of Mechanics, 62
Diseases of Field and Garden Crops, by Worthington G. Smith,
825
Expansion of England, by Mr J. R. Seeley, Professor of Modern
History, 62
Guide to Methods of Insect Life, and Prevention and Remedy of
Insect Ravage, 271
Introduction to the Study of Modern Forest Economy, by Dr J. C.
Brown, 825
Killin Collection of Gaelic Songs, by Mr Charles Stewart, 824
London Cries; Chap-book Chaplets; and Bygone Beauties,
published by Messrs Field and Tuer, 63
More Bits from Blinkbonny, by ‘John Strathesk,’ 824
Norman Conquest, by Mr William Hunt, 411
Photography for Amateurs, by Mr T. C. Hepworth, 824
Practical Taxidermy, by Montague Brown, F.Z.S., 825
Shetland and the Shetlanders, by Sheriff Rampini, 271
Sprigs of Heather, or the Rambles of ‘Mayfly’ with Old Friends,
by Rev. John Anderson, D.D., 208
Whitaker’s Almanac for 1884, 63
Miscellaneous Articles of Instruction
and Entertainment.
Abe, Story of, 817
Acorns, Under the, 657
Acrobats, 318
Advertisers Again, Among the, 94
Almanacs, Romance of, 23
Amateur ‘Cabby,’ an, 778
America, European Emigration to, and its Effects, 641
American Newspapers on Themselves, 714
Amusements in Germany, Popular, 634
Ancient and Modern Statues, the Largest, 470
Ancient People, an, 430
Anglo-Indian Chaplain, Recollections of an, 792
Animal Life, Studies in, 822
—— Memorials and Mementoes, 285
Antipathies in Animals—
i. Horses, 85

ii. Dogs, 590

Architecture, Stained Glass as an Accessory to Domestic, 359
Army Schools, 494
Arsenic in Domestic Fabrics, 799
Artificial Jewels, 731
Ashburnham Collections, the, 341
Back from ‘Eldorado,’ 573
Bank of England, Curiosities of the, 737
Bird Migration, 481
Birds of Spring, 129
Bonded Warehouses, London, 58
Break-neck Venture, a, 588
Bridge, the Haunted, 814
British Museum, New Mediæval Room at the, 693
Brompton Cemetery, In, 753
Buried Alive, 222
Bushranger, Interviewed by a, 650
‘Cabby,’ an Amateur, 778
Calls before the Curtain, 135
Cameo-cutting, 224
Cave-chapels, 513
Charr of Windermere, the, 406
Chewton-Abbot, 280, 295, 315
Children, Over-educating, 366
Christmas Trees, 748
Cigars, 709
Circulating-library Critics, 81
Cliff-houses of Cañon de Chelly, 40
Coin Treasures, 249
Coins Wearing Away? Are our, 393
Colds, Common, 175
College, Queen Margaret, 555
—— Rooms, My Old, 262
Colonel Redgrave’s Legacy, 780, 793, 811
Colour-sense, 44
Commercial Products of the Whale, 566
Conversation, the Art of, 442
Cooking Classes for Children, 775
Cooking-stoves, Gas, 367
‘Corners,’ 289
Correspondence Classes, 555
Cricket, Umpires at, 399
Curiosities of the Bank of England, 737
Curiosities of the Electric Light, 140
—— —— —— Microphone, 373
—— —— —— Peerage, Some, 305, 326
Curiosity in Journalism, a, 200
Cycling, Progress of, 335
Cyprus Locusts, 801
Dauphins, False, 662
Death-claims, How Life-offices pay their, 97
Decisions, Some Legal, 423
Deer-forests, Scottish, 721
Detective Police, Our, 337
Dinner-parties Out of Doors, 673
Dishes, Some Queer, 230
Distillation in Ireland, Illicit, 644
Dwarfie Stone, Legend of the, 667
Eastern Trading, Some Instances of, 463
Edicts, Ancient Rock-hewn, 486
Educational Pioneer, an, 699
‘Eldorado,’ Back from, 573
Electric Light, Curiosities of the, 140
Electricity and Gas, the Future of, 625
—— for Nothing! 453
——, Lighting Collieries by, 496
Elephants, the Moulmein, 638
——, Trimming the Feet of, 240
Emigration to America, European, 641
English Law, Familiar Sketches of—
Marriages; Settlements; and Breaches of Promise to
i.
Marry, 102
ii. Parent and Child, 377
iii. Master and Servant, 490
Episodes of Literary Manuscripts, 283
Erratic Pens, 313
Errors in Domestic Medicine, Common, 299
European Emigration to America, and its Effects, 641
Explosion, Story of a Vast, 705
Fairs, Old Provincial, 598
Falkland Islands, a Peep at the, 110
False Dauphins, 662
Fellow-passenger, My, 252, 263
Florida, Concerning, 797
Food-notes, Some, 287
Foresight of Insects for their Young, 587
Forestry and Farming, 720
—— Exhibition, Edinburgh, 1884, International, 193
Fortunes, Sudden, 241
French Detectives, 48
Frendraught, the Fire of, 52
Fuel, a New, 671
Furniture Saleroom, In a, 379
Gas Cooking-stoves, 367
——, the Future of Electricity and, 625
Gentleman of the Road, a, 429
Germany, Popular Amusements in, 634
Girls, Wives, and Mothers, 33
Glacier Garden, a, 785
Gold, 209
Gold-fields, the Transvaal, 177
Good-natured People, Mischief done by, 111
Gordon’s College, Aberdeen, 183
‘Grand Day,’ 561
Greenroom Romance, a, 471
Grouse, 529
Gum-arabic and the Soudan, 640
Hampstead Heath, 65
‘Happy Ever After,’ 161
Haunted Houses, Rationale of, 397
Health, Our, 113, 234, 401
Heroines, 492
Highland Glen, In a, 511
Holiday, a River, 545
‘Home! Sweet Home!’ 173
Home-nursing, 417, 549, 609, 725
Homing Pigeon, the, 245
Honey-bee, Something about the, 409
Hospitals and Dispensaries, London, 518
Housewives, Hints for, 447
Humorous Definitions, 475, 669
Hush-money, 143
In a Flash, 520
India, Musk-rat of, 703
Indian Jugglers, 604
—— Snakes, 214
Insects, Foresight of, for their Young, 587
Ireland, Illicit Distillation in, 644
Island, a Solitary, 719
——, an Interesting, 347
Jaffa to Jerusalem, From, 321
‘Jerry-building’ in the Middle Ages, 464
Jewels, Artificial, 731
Joint-stock Companies and ‘Limited Liability,’ 577
Journalism, a Curiosity in, 200
‘King Country, the,’ 637
—— of Acres, a, 12, 29
‘Kitchen Kaffir,’ the, 117
Knowledge, a Little, not Dangerous, 616
Last of the Stuarts, the, 600, 617
Law, Sketches of English, 102, 377, 490
Legal Decisions, Some, 423
Life, Prolonging, 427
Life-assurance and Annuities, Post-office, 257
Lifeboat Competition, 459
Life-offices, How they pay their Death-claims, 97
Lighting Collieries by Electricity, 496
‘Limited Liability,’ Joint-stock Companies and, 577
Literary Beginners, Another Word to, 49
Literary Manuscripts, Episodes of, 283
—— Self-estimates, 220
Locusts, Cyprus, 801
London Bonded Warehouses, 58
—— Hospitals and Dispensaries, 518
London, Nature around, 225