MicroRNAs as potential clinical biomarkers:

emerging approaches for their detection

SK Srivastava1, A Bhardwaj1, SJ Leavesley2,3, WE Grizzle4, S Singh1, AP Singh1,5
1Mitchell Cancer Institute, University of South Alabama, Mobile, 2Department of Chemical and Biomolecular Engineering,

University of South Alabama, Mobile, 3Department of Pharmacology, University of South Alabama, Mobile,
4Department of Pathology, University of Alabama at Birmingham, Birmingham, and 5Department of Biochemistry

and Molecular Biology, University of South Alabama, Mobile, Alabama

Abstract
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MicroRNAs (miRNAs) have emerged as novel post-transcriptional regulators of gene expression.

These short non-coding RNAs are involved in diverse biological processes and their dysregulation
is often observed under diseased conditions. Therefore, miRNAs hold great potential as clinical
biomarkers of physiological and pathological states and extensive efforts are underway to develop
efficient approaches for their detection. We review recent advances and discuss the promises and
pitfalls of emerging methods of miRNA profiling and detection.

Key words: biomarkers, clinical applications, detection, microRNAs, miRNAs

For personal use only.

MicroRNAs (miRNAs) are small noncoding 19–25 Alzheimer ’s disease (Hebert and De 2009) and
base pair RNAs that act as post-transcriptional many human malignancies (Ventura and Jacks
regulators of gene expression (Bhardwaj et al. 2010, 2009, Visone and Croce 2009). miRNA expression
Janga and Vallabhaneni 2011). A single miRNA is developmentally and physiologically regulated,
can target multiple mRNAs and conversely, a and an aberrant expression of miRNAs is often
single mRNA can be targeted by several differ- detected under pathological conditions (Cheng
ent miRNAs (Pillai 2005, Sethupathy et al. 2006). et al. 2007, Noh et al. 2009, Pauley et al. 2008,
To date, several hundred miRNAs have been Roldo et al. 2006). Because of the functions of dif-
identified in humans and their number continues ferent miRNAs and their differential expression
to increase, which indicates that miRNAs are a patterns under disease conditions, miRNAs are
major component of the gene regulatory system emerging as a novel class of clinical biomarkers
(Bhardwaj et al. 2010). miRNAs are evolutionarily for diagnosis, prognosis, disease classification,
conserved regulatory molecules and evidence is and monitoring of progression and response
accumulating that suggests their involvement to therapeutic interventions. Therefore, several
in a variety of normal and pathophysiological novel methods have been developed and many
processes (Lewis et al. 2005, O’Driscoll 2006). are still emerging to achieve better sensitivity,
miRNAs play significant roles in diseases includ- specificity and high throughput under clinical
ing diabetes (Poy et al. 2004), heart diseases (Cheng settings. We review here some of the findings
et al. 2007, Van et al. 2006), neurological disorders that suggest the clinical potential of miRNAs as
such as fragile X syndrome (Caudy et al. 2002), biomarkers and discuss emerging methods for
miRNA profiling.

miRNAs as clinical biomarkers

Correspondence: Ajay P. Singh, Ph.D., Department of Oncologic
Sciences, Mitchell Cancer Institute, University of South Alabama, Biomarkers are specific features that can be used
1660 Springhill Avenue, Mobile, AL 36604-1405. Tel: 251-445-
9843; Fax: 251-460-6994. E-mail: [email protected]
as indicators of biological processes. With recent
© 2013 The Biological Stain Commission scientific and technological advancements, newer
Biotechnic & Histochemistry 2013, Early Online: 1–15. biomarkers have been identified that are more

DOI:10.3109/10520295.2012.730153 1
 sensitive, more specific and whose detection is cells of patients suffering from rheumatoid arthritis
feasible in a clinical setting. There is much in the (Pauley et al. 2008). Nakasa et al. (2008) reported
literature that indicates the potential of miRNAs increased expression of miR-146a in different cells
as clinical biomarkers of disease progression, dif- and tissues of rheumatoid arthritis patients and
ferential diagnosis, therapy response and molecu- suggested that it is associated with the severity
lar typing (Andorfer et al. 2011, Cacchiarelli et al. of the disease. Kong et al. (2011) investigated the
2011, Saito et al. 2011, Scapoli et al. 2010, Grizzle expression of several miRNAs in newly diagnosed
et al. in press, Manne et al. 2010) (Table 1). miRNAs type 2 diabetic (nT2D) and pre-diabetic patients and
have the advantage of greater stability compared observed that seven miRNAs, i.e., miR-9, miR-29a,
to other nucleotide based markers. miRNAs can miR-30d, miR-34a, miR-124a, miR-146a and miR-
be detected easily in tissues, body fluids and even 375, were elevated significantly in nT2D compared
formalin fixed, paraffin embedded (FFPE) tissue to pre-diabetes. Kapsimali et al. (2007) used miRNA
samples (Cortez and Calin 2009, Nelson et al. 2006a, profiling to characterize neural cells, which might
Bovell et al. 2012). For example, elevated levels of facilitate the prediction of likely modes of miRNA
miR-142-3p were detected in the sera of patients function in the central nervous system. Also, a
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with systemic sclerosis and these levels were cor- specific miRNA expression was observed during the
related with the severity of the disease (Makino early phase of acute myocardial infarction (Dong
et al. 2011). Enhanced expression of miRNAs, such et al. 2009).
as miR-155, miR-146a, miR-132 and miR-16, also Several groups have exploited the potential
has been detected in peripheral blood mononuclear of using miRNAs for discriminating cancer from

Table 1. List of selected miRNAs and their clinical association

miRNA Disease Clinical association References

For personal use only.

miR-142-3p (↑) Systemic sclerosis Severity of disease Makino et al. 2011

miR-155 (↑), miR-146a (↑), Rheumatoid arthritis Pathogenesis of disease Pauley et al. 2008
miR-132 (↑), and miR-16(↑)
miR-9 (↑), miR-29a (↑), Newly diagnosed type 2 Pathogenesis of disease Kong et al. 2011
miR-30d (↑), miR-34a (↑), diabetic
miR-124a (↑), miR-146a (↑)
and miR-375 (↑)
miR-103 (↑), miR-107 (↑), Pancreatic cancer Distinguish tumors with Roldo et al. 2006
miR-155(↓), miR-21(↑) different clinical behavior
miR-150 (↓) Pancreatic cancer Pathogenesis of disease Srivastava et al. 2011
miR-141 (↑) Prostate cancer Distinguish cancer patients Mitchell et al. 2008
from healthy ones
miR-146a (↓) Prostate cancer Development of hormone Lin et al. 2008
refractory prostate
cancer
miR-10b (↑) Prostate cancer Biochemical relapse of Fendler et al. 2011
disease
miR-10b (↑) Pancreatic cancer Aggresive behavior of Nakata et al. 2011
disease and poor
survival of patient
miR-451 (↓) Gastric cancer Survival of patient Bandres et al. 2009
miR-34a (↓) Chronic lymphocytic leukemia Drug resistance Zenz et al. 2008
miR-196a (↓) Barrett’s esophageal Disease progression Maru et al. 2009
metaplasia-dysplasia-invasive
adenocarcinoma
miR-122 (↓) Hepatocellular carcinoma Metastatic behavior of Coulouarn et al. 2009
disease
miR-145 (↓), miR-21(↑), Bladder cancer Disease progression Dyrskjot et al. 2009
miR-129 (↑)
miR-221 (↓) Prostate cancer Aggressive and metastatic Spahn et al. 2010
behavior and disease

(↑) Up regulated; (↓) Down regulated

2 Biotechnic & Histochemistry 2013, Early Online: 1–15

 benign diseases and normal conditions (Bloomston pancreatic ductal cells and overexpression of
et al. 2007, Osaki et al. 2008, Roldo et al. 2006). miR-10b was significantly associated with poor
Correlation of miRNA expression with cancer type, prognosis of patients with pancreatic cancer.
tumor stage and response to drug treatments also Bandres et al. (2009) demonstrated that down-
has been reported (Lu et al. 2005, Nakajima et al. regulation of miR-451 was linked with prognosis
2006, Nass et al. 2009, Zenz et al. 2008). Roldo et al. in patients with gastric cancer. In chronic lympho-
(2006) suggested that miRNA expression profiling cytic leukemia, reduced expression of miR-34a was
may be useful for distinguishing pancreatic tumors correlated with resistance to chemotherapy (Zenz
with different clinical behavior. These investiga- et al. 2008). Maru et al. (2009) identified miR-196a
tors reported that expression of miR-103 and miR- as a potential marker of progression for Barrett’s
107 associated with lack of expression of miR-155 esophageal metaplasia-dysplasia-invasive adeno-
could discriminate pancreatic tumors from normal carcinoma sequence. In hepatocellular carcinoma,
tissue, while a set of ten other miRNAs could dis- loss of miR-122 expression in tumor cells was
criminate endocrine from acinar tumors. Further- correlated with suppression of the hepatic phe-
more, they showed that miR-204 was expressed notype and development of metastatic properties
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primarily in insulinomas and the overexpression of (Coulouarn et al. 2009). Dyrskjot et al. (2009)
miR-21 was correlated strongly with high Ki67 pro- identified miR-129 as a potential miRNA for pre-
liferation index and occurrence of liver metastasis. dicting disease progression in bladder cancer. In a
We observed recently that miR-150 was expressed large cohort study, miR-221 was determined to be
differentially and exhibited significant down- down-regulated progressively in aggressive forms
regulation in pancreatic cancer tissues compared of prostate cancer and also was associated with
to normal pancreas (Srivastava et al. 2011). In a recurrence of prostate cancer (Spahn et al. 2010).
study by Bloomston et al. (2007), expression pro- All of these studies strongly suggest that miRNAs
filing of miRNAs in pancreatic cancer, normal can serve as useful biomarkers for multiple clinical
pancreas and chronic pancreatitis demonstrated applications (Fig. 1).
For personal use only.

overexpression of 21 miRNAs and down-regulation

of 4 miRNAs in pancreatic cancer, which suggests Emerging approaches for miRNA
their utility for distinguishing pancreatic cancer
profiling and detection
from benign pancreatic tissue. To study the role
of miRNAs in the progression of prostate cancer,
Conventional methods
Lin et al. (2008) profiled miRNA in both prostate
cancer cells and human prostate cancerous tissues. Several approaches that have been used for
They reported that down-regulation of miR-146a detecting nucleic acids also have been employed
was associated with the development of hormone- for miRNA detection including Northern blotting,
refractory prostate cancer. Mitchell et al. (2008) reverse-transcription-polymerase chain reaction,
determined the level of miR-141 in serum from and in situ hybridization. We discuss these assays
patients with prostate cancer and reported that below with the recent modifications that have
the miR-141 level was higher in the serum of cancer
patients; thus differential miRNA expression may
be useful as potential peripheral blood based
Molecular
biomarkers. Fendler et al. (2011) observed that typing/classification
miR-10b was expressed differentially in primary
prostate cancers and normal adjacent tissues
obtained by radical prostatectomy, and therefore
Prognosis
Diagnosis

might have implications in biochemical relapse of MicroRNAs as

prostate cancer. biomarkers
The potential of miRNAs for assessing disease
aggressiveness and patient survival, and for mon-
itoring therapeutic responses also has been inves- Therapy-response
and follow up
tigated (Hummel et al. 2010, Nakajima et al. 2006,
Segura et al. 2010, Yu et al. 2008). Nakata et al. Fig. 1. Potential applications of miRNAs as clinical
(2011) correlated the expression of miR-10b with biomarkers. Based on our current knowledge, several
severity of pancreatic cancer. They observed that clinical applications of miRNAs are proposed as biomarkers
the expression level of miR-10b was significantly of diagnosis, prognosis, therapy response and follow-up,
greater in pancreatic cancer cells than in normal and molecular classification of the disease.

Emerging approaches for microRNA detection 3

 been made to improve their analytical power and real time PCR detection (a miRNA specific forward
applicability. primer and a universal reverse primer recogniz-
ing the identical tail sequence). Shi and Chiang
(2005) modified this method by adding an artifi-
Northern blotting cial poly(A) tail to the miRNAs, which allowed
the use of an anchored oligo(dT) RT primer with
Northern blotting analysis as a technique for
a 5’ tail. This method exhibited greater sensitivity
miRNA analyses has the advantage that it does
and could detect miRNA with only 100 pg of total
not require special equipment and it can be done
RNA. Chen et al. (2005) developed a somewhat dif-
in normal laboratory settings. Moreover, it can be
ferent approach. These investigators observed that
used to detect both mature and precursor forms of
using stem loop RT primers with a 3’ adapter spe-
any miRNA. The procedure involves separation of
cific for each miRNA worked better than the lin-
small RNAs by gel electrophoresis, transfer from
ear RT primer and provided better specificity and
the gel to a membrane, cross-linking of resolved
sensitivity than linear primers. (Chen et al. 2005).
RNAs by fixation followed by hybridization with
Tang et al. (2006) enhanced the assay further by
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labeled probes. One of the major drawbacks of this

combining the use of a stem loop RT primer with
technique is its low sensitivity. Consequently, the
a single tube reverse transcription followed by a
method requires a large quantity of RNA for detec-
single tube pre-PCR. This technique proved very
tion of miRNA, which in most cases, especially for
sensitive and could detect as little as 220 miRNAs
clinical samples, is not possible. Another drawback
from the RNA content of a single embryonic stem
is that it can detect only one miRNA at a time.
cell. Thus, the problems related to the short length
Several improvements have been made in traditional
of mature miRNAs have been overcome and real
Northern blotting protocols to overcome some of
time PCR today represents one of the most sensi-
these limitations. One of the major improvements
tive and convenient miRNA detection methods.
is the use of locked nucleic acid (LNA) probes for
Northern blot detection, which can increase the
For personal use only.

sensitivity of the assay by about 10-fold (Valoczi In situ hybridization

et al. 2004, Varallyay et al. 2007). In addition,
In situ hybridization (ISH) is widely used for
Ramkissoon et al. (2006) have developed an improved
miRNA detection. The merit of this technique is
method that uses digoxigenin labeled RNA probes
that it can be used to determine the subcellular
for miRNA detection. Incorporation of digoxigenin
distribution of miRNA (Kloosterman et al. 2006,
increases the shelf-life of the probes and eliminates
Nelson et al. 2006b, Wienholds et al. 2005). How-
the radioactivity hazard. This assay remains time-
ever, conventional ISH is not efficient for miRNA
consuming, however, and requires larger samples,
detection in tissues. The major challenge is fixing
so it is not suitable for clinical settings.
small RNAs to prevent their loss during hybrid-
ization or washing procedures so that inconsistent
or false negative results can be avoided. Further-
Reverse transcription-polymerase chain
more, it is more tedious compared to other miRNA
reaction (RT-PCR)
profiling techniques and also is low throughput.
Polymerase chain reaction (PCR) based methods Despite these drawbacks, the use of locked nucleic
have the advantage of being highly sensitive. With acid (LNA) probes (Kauppinen et al. 2006, Valoczi
technical advancements, it is now possible to detect et al. 2004) enhanced the specificity and sensitiv-
low copy numbers of both the precursor and the ity of miRNA detection by ISH and the technique
mature forms of miRNAs with great sensitivity has been employed in zebra fish, mice, and frog
and specificity (Chen et al. 2005, Jiang et al. 2005, embryos (Kloosterman et al. 2006, Wienholds et al.
Schmittgen et al. 2004). Schmittgen et al. (2004) 2005). Nelson et al. (2006b) modified this method
were the first to determine miRNA expression using the miRNA microarray platform RAKE
by RT-PCR, but the method could not detect the (RNA-primed, array based, Klenow Enzyme)
mature form of the miRNAs owing to their small together with LNA based in situ hybridization.
size. Raymond et al. (2005) subsequently designed This enabled detection of miRNA in FFPE tissues
a primer for a reverse transcription (RT) primer from human brain and oligodendroglia tumors.
complementary to the 3’ end of the mature miRNA Deo et al. (2006) improved the specificity further
that also contained a 5’ tail sequence. This created by using fluorescein hapten-linked RNA oligonu-
a template that was longer than the mature miRNA cleotide probes and modified washing conditions.
for which specific primers could be designed for Another improvement, which combined the use of

4 Biotechnic & Histochemistry 2013, Early Online: 1–15

 LNA with tyramide signal amplification, resulted of the invader assay can be increased by adding an
in rapid and efficient detection of miRNAs in fro- arrestor oligonucleotide probe that maintains low
zen tissues (Stenvang et al. 2008). This method background. The arrestor probe hybridizes with the
obviates the need for microdissecting malignant uncleaved probe, thus not allowing it to participate
cells when these are mixed with inflammatory and in the secondary reaction (Fig. 2B). This assay is
stromal cells in specific tumors. rapid and has high-throughput efficiency. Further-
more, it is extremely sensitive, which allows detec-
tion of miRNAs from 50 ng of total RNA (Allawi
Emerging methods et al. 2004).
With technological advancement and increasing
interest, novel analytical approaches for profiling Bioluminescence based methods
miRNA expression continue to emerge. Many of
Cissell et al. (2008a) used a bioluminescence reso-
these evolving methods are based on nanotech-
nance energy transfer (BRET) based method for
nology principles and address the limitations of
detecting nucleic acids. BRET is a modified form
conventional methods for clinical applicability by
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of Förster resonance energy transfer (FRET) in

minimizing the sample size required, and increas-
which a bioluminescent molecule is substituted
ing sensitivity, precision and dynamic range,
for the donor fluorophore. Although this method
while at the same time being fast and less labor-
was applied first by Xu et al. (1999), these inves-
intensive.
tigators appropriately point out that some of the
original BRET observations were made during
the isolation of aequorin by Shimomura et al.
Invader assay
(1962), because aequorin-GFP compose a BRET
The invader assay developed by Allawi et al. (2004) pair (Johnson et al. 1962). For nucleic acid assays,
can discriminate between precursor and mature Cissell et al. (2008a) used Renilla luciferase (Rluc),
For personal use only.

miRNAs as well as between the target miRNA and a protein that emits bioluminescence in the pres-
its closely related isotypes. The assay combines ence of the substrate, i.e., coelenterazine, as the
enzyme based cleavage and fluorescence based bioluminescent donor and a red emitting quantum
reactions in the same tube and is based on a prin- dot (QD705) as an acceptor molecule. Rluc has a
ciple similar to that described earlier for the invader bioluminescence emission peak near 480 nm (Xu
mRNA assay (De et al. 2002, Eis et al. 2001). It uses et al. 1999), while QD705 has the characteristic
two different oligonucleotide probes: one “probe fluorescent nanocrystal absorption spectrum with
oligonucleotide” and an “invasive oligonucleotide”; high absorption from 300–490 nm (Invitrogen, Life
the probes are modified by including 2’-O-methy- Technologies, Grand Island, NY). QD705 emis-
lated stem loops at their 5’ and 3’ ends, respectively, sion occurs at 702 nm. Thus, Rluc-QD705 forms a
to stabilize hybrids containing the target RNA BRET pair with significant spectral overlap and a
stacked between the two short base pair duplexes. very large Stokes’ shift; the former provides high
The invasive oligonucleotide has a 3’ terminal over- energy transfer efficiency and the latter facilitates
lapping nucleotide that is not complementary to spectral separation of Rluc and QD705 emission.
the miRNA sequence. The probe oligonucleotide Because resonance energy transfer efficiency is
has a 5’ flap sequence that is noncomplementary to dependent to the 6th power on the donor-emitter
the miRNA sequence. During assay, both of these intermolecular distance, the BRET assay is highly
probes hybridize to the respective complementary sensitive to coupling of Rluc to QD705.
sites of the miRNA, which leaves a 5’ flap in the The BRET nucleic acid assay uses an amine-
probe oligonucleotide (Fig. 2A). A structure-specific modified oligonucleotide probe specific to the
5’ nuclease cleaves the 5’ flap sequence from the target nucleic acid and is labeled with a carboxy-
probe oligonucleotide, which then acts as an inva- activated QD705. A separate, partially complemen-
sive oligonucleotide for the 3’ end of a secondary tary, competitive probe also was created by the
reaction template (SRT). A new probe that contains addition of a thiol group and labeled with Rluc.
a fluorophore and quencher hybridizes to the 5’ The QD705 and Rluc probes hybridize with each
end of the SRT in a way that slightly overlaps the other upon mixing and generate a BRET signal
flap sequence. This promotes a second cleavage in a at 702 nm caused by the energy transfer from
similar way by 5’ nuclease and separates the fluoro- Rluc to QD705 in the presence of coelenterazine.
phore from its quencher, thus generating a fluores- The presence of the nucleic acid target decreases
cent signal that can be detected (Fig. 2A). Sensitivity the BRET signal by competitive hybridization

Emerging approaches for microRNA detection 5

 C
A
G
T CTGAA--
5' --ATCGA
(A) Invasive --UAGCGACUU--
probe Probe

Target miRNA

Released Flap
SRT
Q
F
FRET probe
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F Q
FRET probe
(B)
Uncleaved probe

F Released Arrestor probe

fluorophore
Fluorophore
For personal use only.

miRNA copy number

Data generation
and analysis

Fig. 2. Invader assay. A) The assay involves two reactions. In the primary reaction, invasive and probe oligonucleotides
with hairpin overhangs for enhancing stability are bound to the target miRNA. Hybridization results in the formation of a
5’ overlap-flap structure from the probe oligonucleotide, which serves as a substrate for the structure-specific 5’ nuclease,
which causes release of the 5’ flap. In the secondary reaction, a FRET oligonucleotide labeled with a fluorophore (F) and
a quencher (Q) is added to a secondary reaction template (SRT). The released 5’ flap from the primary reaction acts as
an invasive probe in the secondary reaction, which leads to the formation of a second fluorophore-conjugated overlap-flap
structure. Cleavage of this 5’ flap releases the fluorophore to generate a signal that can be quantified. B) An arrestor
oligonucleotide complementary to the probe oligonucleotide is used in the secondary reaction that binds to the uncleaved
probe to avoid its interference in the binding of the FRET probe to SRT, which reduces the background noise.

with the QD705 probe, which inhibits Rluc-QD705 biotinylated anti-miRNA probe that is immobilized
proximity (Fig. 3A) (Cissell et al. 2008a). One pos- on a neutravidin-coated microtiter plate (Fig. 3B).
sible limitation of this technique is the reduced The unlabeled miRNA and Rluc-labeled miRNA
emission intensity of bioluminescence assays com- compete to hybridize with the immobilized bioti-
pared to fluorescence assays. Therefore, a high nylated anti-miRNA probe. After hybridization,
sensitivity detection system is necessary for this unbound miRNAs are removed by washing and
technique and high speed assays may be difficult Rluc substrate, i.e., coelenterazine, is added. The
to implement. resulting bioluminescence may be measured by
Cissell et al. (2008b) also modified the earlier standard radiometric or spectroscopic techniques.
BRET method by developing a competitive oligonu- The decrease in bioluminescence emission is cal-
cleotide hybridization assay, which uses only Rluc culated and related to the concentration of target
as a direct label for miRNA detection. This method miRNA using a calibration curve (Fig. 3B). There-
overcomes the need for a dual reporter assay, which fore, this assay permits a simplified detector config-
simplifies the detection requirements. The method uration with very high sensitivity, as little as 1 fmol
requires a combination of unlabeled synthetic detection limit, and it can be used for miRNA detec-
target miRNAs, Rluc-labeled target miRNAs and a tion and quantification from crude samples such as

6 Biotechnic & Histochemistry 2013, Early Online: 1–15

 (A) (B)

Neutravidin-
Biotinylated coated plate
QD-probe Rluc-probe miRNA probes
Rluc
Target Unlabeled labeled
nucleic acid miRNA miRNA

Hybridization Competitive hybridization

Competitive hybridization

Coelenterazine

Coelenterazine
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BRET signal No BRET signal

Emitted light
Copy number of miRNA
Data generation
and analysis
For personal use only.

Fig. 3. Bioluminescence based assay. A) Quantum dot labeled oligonucleotide probe (QD-probe) and Rluc labeled
oligonucleotide probe (Rluc-probe) are used in BRET based detection. In the absence of target nucleic acid, QD-probes
hybridize with Rluc-probe to generate a signal after addition of coelenterazine. When the target is present, the BRET
signal is decreased owing to the competitive inhibition of Rluc hybridization with QD-probe. B) In a modified assay,
biotinylated anti-miRNA probes first are immobilized on neutravidin-coated microtiter plate, then Rluc-labeled target
miRNAs and unlabeled synthetic target miRNAs are added. Both sample (Rluc labeled) and synthetic (unlabeled) miRNAs
compete with each other during hybridization with the immobilized probes. Upon addition of coelenterazine, a decrease
in signal intensity is measured as an estimate of target miRNA levels.

blood and saliva. The assay can be performed using the amount of a specific cRNA in the sample (Yang
a 96- or 384-well plate, which offers potentially high et al. 2001). This is an inexpensive, rapid and high
throughput capabilities that could prove extremely throughput method that can detect large numbers
useful in clinical settings. of targets in a single experiment. Lu et al. (2005)
later modified the BADGE method to enable accu-
rate and inexpensive miRNA profiling in which
Bead based flow cytometry
beads conjugated to specific probes were labeled
Yang et al. (2001) developed a flow cytometry with a mixture of two different fluorescent dyes.
based “beads array for detection of gene expres- These investigators first attached adaptor mol-
sion” (BADGE) method for analysis of expres- ecules at both the 5’ and 3’ ends of miRNA and RT
sions of multiple genes. These investigators used was performed using primers complementary to
fluorescent microspheres conjugated to capture the adaptors. Following RT, miRNA amplification
probes that are specific for target cRNA that had was performed using biotinylated primers, then
been labeled by incorporating biotinylated UTP. hybridized to the capture probes complementary to
The mixture of different beads conjugated with the miRNAs. The carboxylated polystyrene beads
capture probes was allowed to hybridize with permeated with variable mixtures of two different
the labeled target sample followed by addition of fluorescent dyes can generate up to 100 colors; each
streptavidin-conjugated R-phycoerythrin to allow color represents a single miRNA. After hybridiza-
its binding to the microspheres/capture probe/ tion, these conjugated beads were stained with
cRNA complex. Subsequently, flow cytometry was streptavidin-phycoerythrin and analyzed using a
performed using the Luminex100 system to detect flow cytometer. The bead color measured by flow

Emerging approaches for microRNA detection 7

 cytometry represented a particular miRNA, and that displayed a high cross-correlation coefficient,
intensity of phycoerythrin indicated the abundance the investigators were able to reduce greatly the
of miRNA (Lu et al. 2005). noise of the detection system to achieve great
specificity. Based on this analysis, the investiga-
tors were able to detect coincident events between
Fluorescence correlation spectroscopy (FCS)
the two fluorescent probes as measured at the two
FCS estimates the concentration fluctuations of flu- locations corresponding to the foci of the 532 and
orescent particles in solution by measuring small 633 nm lasers.
fluctuations in fluorescence intensity over time One merit of this assay is that it does not require
(Magde et al. 1974). FCS measurements typically reverse transcription or amplification; rather it is
are made within a small interrogation volume that based simply on hybridization of two spectrally
is determined by the spot size of a laser beam in distinguishable fluorescent oligonucleotide LNA
a confocal microscope. Because of the great spa- probes. A second merit is that fluorophore signals
tial resolution, FCS measurements can be used to from hybridized probes are counted directly on
measure subcellular diffusion and reaction rates a single molecule level, which yields a detection
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in living cells (Kim et al. 2007). Therefore, FCS limit of 500 fmol miRNA. This method has great
is an accurate and sensitive approach to study- potential owing to its great sensitivity, speed, and
ing molecular interactions under physiological ability to differentiate single base mismatches.
conditions (Hui et al. 2010). Using this method, the Using this technique, Neely et al. (2006) measured
fluorescence emitted from a relatively small num- the expression profile of 45 different miRNA tar-
ber of fluorophores within the interrogation vol- gets in 16 different human tissues from very small
ume can be measured over time. These fluctuations amounts of total RNA.
in fluorescence are analyzed using autocorrelation One potential disadvantage of this assay is that
techniques, the results of which subsequently are it uses a highly customized combination of micro-
fitted to a model that describes the diffusion and fluidics and single photon counting detection that
For personal use only.

reaction characteristics of the system (Kim et al. currently is not available as a stand alone system.
2007). This technique has been used to quantify It may be possible to translate this approach to
properties of single molecules in solution. By using single FCS systems, but this likely will introduce
a multicolor confocal system, it is possible to ana- a great reduction in sensitivity and a great increase
lyze the interactions of many molecules, in which in noise, because single molecule counting will
case the technique commonly is referred to as not be measurable directly as in the microfluidic
fluorescence cross-correlation spectroscopy (Bacia flow channel used by Neely et al. (2006). Finally,
et al. 2006). although this approach did not require the full
Another flow cytometry based assay for miRNA auto-correlation analysis and fitting to physical
measurement has been described by Neely et al. models that is employed in traditional FCS tech-
(2006). In this approach, single molecules were niques (Kim et al. 2007), a basic understanding
counted by employing a microfluidics system, laser of molecular transport and reaction models still
excitation and avalanche photodiode detection in is encouraged when considering this approach
an optical configuration similar to that employed (Kim et al. 2007, Magde et al. 1974).
by many flow cytometers. Two spectrally dis-
tinguishable fluorescent probes were used, each
Surface-enhanced Raman spectroscopy (SERS)
complementary to half the target sequence of
the miRNA of interest. A quencher containing an Raman spectroscopy is a highly specific method
oligonucleotide sequence complementary to the for molecular characterization, because the inelastic
unbound labeled probes also was used to decrease light scatter signal that is generated during Raman
the background noise. Two-color laser excitation scattering depends on the vibrational states of the
(532 and 633 nm) was used with laser foci stag- molecule. Raman spectra typically provide many
gered along the length of the flow channel to allow narrow peaks that can be used to infer much about
sequential excitation of each fluorophore. In addi- the structure of a molecule. These narrow peaks
tion, foci from two lasers at identical wavelength also can be used to differentiate similar molecules
were staggered along the length of the flow chan- based on statistical clustering or decomposition
nel. Triggering was performed post-acquisition techniques, often with great specificity. Unfortu-
based on the cross-correlation of fluorescence emis- nately, traditional Raman scattering produces very
sion owing to the excitation of these two identical weak signals, roughly 1010 times weaker than fluo-
wavelength lasers. By restricting analysis to events rescence signals. Because of this, traditional Raman

8 Biotechnic & Histochemistry 2013, Early Online: 1–15

 spectroscopy has been limited in terms of sensitivity their ability to identify correctly the composition
and signal acquisition times. SERS is a surface- of miRNA mixtures. Finally, miRNA detection also
sensitive technique that enhances the Raman scat- may be complicated by the background signals
tering signal by performing measurements in the in complex (biological) samples that are gener-
presence of a rough metallic surface. The interaction ated by SERS. Nevertheless, SERS is an extremely
of laser illumination with the metallic surface cre- sensitive, accurate, and powerful method for
ates surface plasmons, which greatly magnify the identification and classification of miRNAs and
Raman signal. SERS signal intensities can be on the could be employed for routine miRNA expression
order of 106 times greater than those of unenhanced profiling, after appropriate sample purification
Raman signals (Kneipp 2007). (Driskell et al. 2008).
SERS has been used successfully as a potential
miRNA detection technique by Driskell et al. (2008).
Surface plasmon resonance imaging (SPRI)
These investigators detected enhanced Raman sig-
nals from single species of miRNA when blotted SPRI is an imaging technique in which surface plas-
onto a silver nanorod array substrate on a glass mons are excited in a thin metallic surface (Pattnaik
Biotech Histochem Downloaded from informahealthcare.com by Nyu Medical Center on 05/17/13

slide. The silver nanorod substrate, a key element 2005, Steiner 2004). Although surface plasmons can
of their detection approach, was created using be excited by other phenomena, such as electron
oblique angle vapor deposition fabrication, which beams, excitation in microscopy systems usually
increased the substrate reproducibility (Driskell is achieved using a polarized light source, often a
et al. 2008). After substrate preparation, the inves- laser, combined with an attenuated total reflection
tigators spotted different species of miRNA onto coupler, or prism (Steiner et al. 1999). P-polarized
the silver nanorod surface. After adsorption of the light that is incident to the surface excites surface
miRNAs, the SERS spectrum then was obtained plasmons within a narrow range of incident angles
for each species. Multiple measurements from (Steiner 2004). A charge-coupled device camera
each spot and multiple spots for each miRNA then is used to detect the reflected light. Because
For personal use only.

species were assessed to test the reproducibility of the polarization coupling requirement, p-polar-
of this approach. Driskell et al. (2008) found that ized light is used to visualize surface absorption,
the spectra generated for different miRNAs were while s-polarized light typically is used as a refer-
similar with regard to the number and location ence image. Surface plasmon excitation is highly
of the scattered bands owing to the presence of dependent on the thickness and refractive index
similar nucleotides. The relative intensities of the of the local environment. These parameters can be
respective bands, however, were different for each influenced by subtle changes in the local environ-
miRNA species, because the relative number and ment, such as ligand binding or intermolecular
composition of nucleotides varied among species. interactions, which makes SPRI a highly sensitive
Furthermore, these investigators demonstrated molecular detection technique.
that distinct features of the SERS spectrum changed SPRI recently has been employed for miRNA
linearly with changes in the nucleotide content, profiling by Fang et al. (2006). These investiga-
which indicates that the approach could be applied tors developed an amplification approach that
to gain information from unknown miRNA spe- enables miRNA detection with very great sen-
cies. They then used a partial least squares regres- sitivity, which was combined with patterning
sion approach to demonstrate that miRNA species of LNA binding regions on the surface of a thin
could be identified accurately based on their SERS film to enable detection of a panel of miRNAs. To
spectrum. They also demonstrated that five differ- develop this assay, the investigators immobilized
ent miRNAs in a sample set could be discriminated LNA probes that were partially complementary
accurately using the SERS spectrum. to the target miRNA, leaving a six-nucleotide
The merit of the SERS technique described region, onto an SPRI substrate, presumably gold.
above lies in its excellent reproducibility and single- Hybridization was effected so that miRNAs were
nucleotide specificity. Theoretically, this technique left with a 3’ overhang (non-complementary
could be used to identify and discriminate many region to the LNA probes) to which poly(A) tails
different species of miRNAs; however, this approach were added by poly(A) polymerase. Enhancement
has yet to be demonstrated effectively with mix- of the SPRI signal was achieved by subsequent
tures of miRNAs. Because the peak wavenumbers binding of poly(T) coated gold nanoparticles,
for the bands are identical and only the relative which bound to the poly(A) tails. This increased
heights of each peak vary with miRNA species, the change in the SPRI reflectance image and
linear decomposition methods may be limited in improved the sensitivity of detection (Fig. 4). This

Emerging approaches for microRNA detection 9

 AAAAAAAAA

AAAAAAAAA
3' 3'
3'

T30 coated Au
nanoparticles

5' 5' Poly A tail

Hybridization
Immobilized probes
on Gold/Silver SPRI
Data generation
substrate
and analysis

Fig. 4. SPRI based method. Probes partially complementary to the target miRNA are immobilized on a metal based
surface (gold/silver SPRI surface). Target miRNA hybridizes with the probes leaving a 3′ overhang. Subsequently, a poly(A)
tail is added onto the 3′ overhang and later poly(T) coated gold nanoparticles are adsorbed onto the poly(A) tails and
Biotech Histochem Downloaded from informahealthcare.com by Nyu Medical Center on 05/17/13

detected with SPRI.

technique promises to be a highly sensitive method extremely sensitive detection method, which
for developing panels for miRNA profiling, because could quantify less than a 10 amol concentra-
the authors demonstrated a detection limit of tion of miRNA. These investigators used a Fe-Ru
10 fmol (Fang et al. 2006). One possible limitation of redox pair as a reporter and used an amplification
this technique is that SPRI is highly dependent on scheme based on a nano-structured electrode plat-
changes in temperature (Steiner 2004), which could form. After hybridization of miRNA with peptide
complicate the development of a portable miRNA nucleic acid capture probes, Ru3⫹ was reduced
For personal use only.

profiling tool. at the nanoelectrode surface and generated sig-

nals. These signals were amplified further by
the ferricyanide-coupled electrochemical reduc-
Electrical detection based methods
tion, which regenerated Ru3⫹ from Ru2⫹. This
Electrical detection based methods are emerg- approach achieved greater sensitivity together
ing as highly specific, rapid and high through- with high specificity for mature miRNA detection
put assays for profiling mature miRNAs. These (Yang et al. 2009). Zhang et al. (2009) developed
methods are based on changes in circuit proper- a label-free, rapid and sensitive direct hybrid-
ties as a consequence of miRNA hybridization. ization assay for miRNA detection using silicon
Gao and Yang (2006) used the electrocatalytic nanowires (SiNW). In this assay, peptide nucleic
nanoparticle tags method, which is based on the acids (PNA) were immobilized on the surface of
use of isoniazid-capped OsO2 nanoparticle tags. the SiNW device that acted as receptor for miRNA
Sodium periodate derivatives were hybridized recognition. Upon incubation of PNA functional-
to oligonucleotide capture probes on the elec- ized SiNW with complementary miRNA targets,
trode. Thereafter, signals were generated that the changes in resistance were measured before
linked periodate-treated miRNAs with the OsO2 and after hybridization and were considered to
nanoparticles, which oxidized hydrazine and be a direct estimate of the concentrations of the
resulted in an electrocatalytic activity at 0.10 V hybridized target miRNA. This method provided
(Fig. 5A). This method proved highly sensitive a detection limit of 1 fmol and could discrimi-
and could detect miRNA at 80 fmol concentra- nate between fully matched and single base mis-
tion (Gao and Yang 2006). Later, these investi- matched miRNA sequences (Zhang et al. 2009).
gators modified their strategy to increase the Peng and Gao (2011) developed a strategy that
sensitivity further by labeling the target miRNA produced great sensitivity with low background.
directly with a redox active and electrocatalytic They first tagged target miRNAs with RuO2 nano-
moiety, Ru(PD)2Cl2 (PD ⫽ 1,10-phenanthroline-5, particles and allowed them to hybridize with
6-dione), which has excellent electrocatalytic PNA probes assembled on a gold electrode. A
activity toward the oxidation of hydrazine (Fig. mixture of aniline/H2O2 then was applied. The
5B); therefore, it permits detection of miRNAs at RuO2 nanoparticles polymerize aniline to form
low concentration, i.e., 20 fmol (Gao and Yu 2007). polyaniline only at hybridized miRNA strands,
Recently, Yang et al. (2009) developed another thus generating a clean background (Fig. 6).

10 Biotechnic & Histochemistry 2013, Early Online: 1–15

 Captured probes
(A) (B)

miRNA Ru(PD)2Cl2
Hybridization labeled miRNA
Biotech Histochem Downloaded from informahealthcare.com by Nyu Medical Center on 05/17/13

OsO2 Nano- N2H4

particles
For personal use only.

N2

N2H 4 N2
miRNA

Control

Data generation
and analysis

Fig. 5. Electrical detection based assay. A) Sodium periodate derivatives of target miRNAs are hybridized to probes
captured on a metallic electrode followed by addition of OsO2 nanoparticles and incubation to allow their ligation
with hybridized miRNAs. Subsequently, electrochemical signals are generated owing to the oxidation of hydrazine.
B) To increase sensitivity, target miRNAs are labeled directly with a electrocatalytic moiety, i.e., Ru(PD)2Cl2 (PD ⫽ 1,
10-phenanthroline-5,6-dione), and hybridized to captured probes. Owing to excellent electrocatalytic activity of
Ru(PD)2Cl2 toward the oxidation of hydrazine, low concentrations of miRNA can be detected using this method.

Perspectives progression and therapeutic outcomes. Interest in

miRNAs has increased greatly and the associated
MiRNAs have received significant attention as a literature is expanding rapidly with reports on
new class of gene regulators and accumulating novel miRNAs, their validated gene targets and
evidence clearly indicates their important roles their expression profiling under various disease
in diverse biological functions. Data emerging conditions. At a similar pace, novel methods for
on miRNA functions and dysregulated expres- miRNA profiling also are emerging that facilitate
sion under various pathophysiological condi- the development of clinically feasible approaches
tions have provided strong support for their for detecting miRNA. Several new assays have
clinical use as functional biomarkers. Their use been developed to overcome the size limitation of
can determine the molecular characteristics of mature miRNAs, sequence similarity among vari-
diseases. Similarly, miRNAs may be used for ous members, and their low occurrence in clinical
early diagnosis of diseases as well as to predict samples. Some of these assays are simple, but not

Emerging approaches for microRNA detection 11

 Acknowledgments

This research was funded by NIH/NCI (CA137513),

PNA probes assembled DOD/US Army (PC0739930, PC093309) and
on electrode USAMCI.
Target miRNA
tagged RuO2
nanoparticles Conflict of interest statement: The authors have no
potential conflict of interest to disclose.

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