Serum MicroRNAs Are Promising Novel Biomarkers
Shlomit Gilad1., Eti Meiri1., Yariv Yogev2, Sima Benjamin1, Danit Lebanony1, Noga Yerushalmi1, Hila
Benjamin1, Michal Kushnir1, Hila Cholakh1, Nir Melamed2, Zvi Bentwich1*, Moshe Hod2, Yaron Goren1,
1Rosetta Genomics Ltd., Rehovot, Israel, 2Division of Maternal Fetal Medicine, Rabin Medical Center, Sackler Faculty of Medicine, Tel Aviv University, Petah-Tiqva, Israel
Background: Circulating nucleic acids (CNAs) offer unique opportunities for early diagnosis of clinical conditions. Here we
show that microRNAs, a family of small non-coding regulatory RNAs involved in human development and pathology, are
present in bodily fluids and represent new effective biomarkers.
Methods and Results: After developing protocols for extracting and quantifying microRNAs in serum and other body fluids,
the serum microRNA profiles of several healthy individuals were determined and found to be similar, validating the
robustness of our methods. To address the possibility that the abundance of specific microRNAs might change during
physiological or pathological conditions, serum microRNA levels in pregnant and non pregnant women were compared. In
sera from pregnant women, microRNAs associated with human placenta were significantly elevated and their levels
correlated with pregnancy stage.
Conclusions and Significance: Considering the central role of microRNAs in development and disease, our results highlight
the medically relevant potential of determining microRNA levels in serum and other body fluids. Thus, microRNAs are a new
class of CNAs that promise to serve as useful clinical biomarkers.
Citation: Gilad S, Meiri E, Yogev Y, Benjamin S, Lebanony D, et al. (2008) Serum MicroRNAs Are Promising Novel Biomarkers. PLoS ONE 3(9): e3148. doi:10.1371/
Editor: Simon Williams, Texas Tech University Health Sciences Center, United States of America
Received May 12, 2008; Accepted August 11, 2008; Published September 5, 2008
Copyright: ? 2008 Gilad et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Rosetta Genomics is a commercial for-profit company, which provided funding for the study. Most key investigators are employees of the company.
Competing Interests: Rosetta Genomics is a commercial for-profit company. Most key investigators are employees of the company. MH receives research and
personal funding from Rosetta Genomics.
* E-mail: email@example.com (AC), firstname.lastname@example.org (ZB)
. These authors contributed equally to this work.
Specific clinical biomarkers have the potential to revolutionize
diagnosis and treatment of various medical conditions, ranging
from abnormal pregnancies to myocardial infarctions and cancer.
In particular, a theme of current cancer research is the quest for
sensitive biomarkers that can be exploited to detect early
neoplastic changes. Ideally, biomarkers should be easily accessible
such that they can be sampled non-invasively. Therefore
biomarkers that can be sampled from body fluids, such as serum
or urine, are particularly desirable. In recent years it has become
clear that both cell-free DNA and mRNA are present in serum, as
well as in other body fluids, and that these CNAs represent
potential biomarkers [1–3]. Accordingly, we considered it likely
that microRNAs might be present in bodily fluids.
MicroRNAs are a recently discovered class of small non-coding
RNAs that regulate gene expression and have a critical role in
many biological and pathological processes [4,5]. In general,
microRNAs are regulated and transcribed like protein coding
genes. Subsequent microRNA biogenesis involves discrete pro-
cessing and transport steps, whereby the active moiety of 20–22
nucleotides is excised from a longer RNA precursor that exhibits
specific hairpin structure. Finally, these 20–22 nucleotides are
incorporated into a composite machinery, which promotes partial
duplex formation between the short RNA and the 39 untranslated
regions (UTR) of targeted mRNAs, resulting typically in mammals
in translational silencing [6,7]. A relevant, though at present
enigmatic, feature of microRNA biology is their remarkable
stability. For example, microRNAs are preserved well in tissue
samples even after formalin-fixation and paraffin-embedding and
can be efficiently extracted and evaluated . Therefore, if
microRNAs are indeed circulating, we hypothesized further that
they should preserve their stability in body fluids. Monitoring the
typically small amounts of CNAs in body fluids requires complex
and sensitive extraction and detection methods, which until now
have been prohibitive either practically or economically .
However, we anticipated that the stability of microRNAs should
allow development of practicable detection methods, such that
they can realistically serve as clinical biomarkers.
Although the total number of microRNAs remains controversial
[4,10,11] and the roles of specific microRNAs are only beginning
to be defined, microRNA expression analyses indicate that diverse
tumors display microRNA expression profiles (for mature and/or
precursor microRNAs) significantly different from normal tissue
[12,13]. Furthermore, microRNAs are emerging as highly tissue-
specific biomarkers [14,15] with potential clinical applicability for
defining the cancer origin of metastases, as we have shown recently
. These data led us to expect that the microRNA abundance
profile of bodily fluids might reflect physiological and/or
pathological conditions and, furthermore, might do so more
PLoS ONE | www.plosone.org1 September 2008 | Volume 3 | Issue 9 | e3148
accurately than an mRNA abundance profile. For mRNA must be
translated into protein to have a biological effect whereas
microRNAs are themselves the active moiety, often influencing
the expression of multiple other genes, and thus likely reflect
altered physiology more directly.
Here we validate that microRNAs are present in body fluids and
moreover, demonstrate that microRNA levels in serum reflect
altered physiological conditions, such as pregnancy.
Materials and Methods
Serum samples were collected from 30 women: 10 in the first
pregnancy trimester (6–12 weeks of gestational age), 10 in the third
pregnancy trimester (34–41 weeks of gestational age) and 10 age-
matched non-pregnant women. Eligibility for the study was limited
to normal uncomplicated singleton pregnancies with no known fetal
malformation. All women provided written informed consent and
the local institutional review board approved the study. 8 ml of
blood was collected from each woman directly into serum collection
tubes (Greiner Bio-one, VACUETTEH Serum Tubes 455071). The
whole blood was allowed to stand for about 1 h at RT before being
centrifuged at 1800 g for 10 minutes at RT. The resultant serum
was aliquoted into eppendorf tubes and stored at 280uC.
About 4 ml of urine samples were collected from each
individual in a urine container. The urine was then aliquoted
into eppendorf tubes and kept frozen at 280uC until it was used
for RNA extraction.
100 ml serum or urine was incubated at 56uC for 1 h with
0.65 mg/ml Proteinase K (Sigma P2308). Two synthetic RNAs
(IDT) were spiked-in as controls before acid phenol:chloroform
extraction and then RNA was ETOH precipitated ON at 220uC.
Next, DNase treatment was performed to eliminate residual DNA
fragments. Finally, after a second acid phenol:chloroform
extraction, the pellet was re-suspended in DDW and two
additional synthetic RNAs were spiked-in as controls.
qRT-PCR platform 
RNA was subjected to a polyadenylation reaction as described
previously . Briefly, RNA was incubated in the presence of
poly (A) polymerase (PAP; Takara-2180A), MnCl2, and ATP for
1 h at 37uC. Then, using an oligodT primer harboring a
consensus sequence, reverse transcription was performed on total
RNA using SuperScript II RT (Invitrogen). Next, the cDNA was
amplified by real time PCR; this reaction contained a microRNA-
specific forward primer, a TaqMan probe complementary to the
39 of the specific microRNA sequence as well as to part of the
polyA adaptor sequence, and a universal reverse primer
complementary to the consensus 39 sequence of the oligodT tail.
qRT-PCR can be used to monitor low microRNA levels
specifically and sensitively
We have developed a proprietary qRT-PCR based platform for
evaluating microRNA levels that detects specifically mature
microRNA molecules (see materials and methods and Figure 1A).
Initially, we validated that our platform is capable of discriminat-
ing between homologous microRNA family members that differ
by only a single nucleotide. Three such microRNAs were
generated synthetically (hsa-let 7a, c & d) and each one subjected
to three independent qRT-PCR reactions, where in each reaction
there were present PCR primers specific to only one of the 3
family members. In most of these reactions, we observed
amplification only of the appropriate family member matching
the specific primer, indicating that this qRT-PCR platform detects
microRNA with accuracy at the single nucleotide level (Figure 1B).
Next, we mixed the three synthetic microRNAs and subjected the
mixture to qRT-PCR in the presence of the let-7d primer-probe
set. In parallel, we took the same amount of synthetic let-7d
microRNA as used in the mixture but subjected it alone to qRT-
PCR. These parallel PCR reactions were repeated two further
times, using reducing concentrations of let-7d synthetic micro-
RNA. We observed that the qRT-PCR amplified let-7d
equivalently whether it was alone or in the presence of
homologous family members at all tested concentrations of let-
7d (Figure 1C). In an additional experiment, we wished to confirm
the capability of our platform to detect specific microRNAs in a
biologically relevant complex background. Decreasing concentra-
tions (from 100% down to 0%) of total RNA extracted from liver
tissue were diluted into total RNA extracted from brain tissue and
0.1 ng of these mixtures subjected to PCR reaction, each reaction
containing primers specific for hsa-miR-122a, a liver specific
microRNA . Hsa-miR-122a was detected, even when only
0.03% of liver RNA was spiked into brain RNA (Figure 1D).
Importantly, hsa-miR-122a detection was linear and there was no
detection in the absence of liver RNA (100% brain RNA).
Finally, to validate the reproducibility of our methods, at two
independent times, two different researchers subjected the same
mix of 32 microRNAs to qRT-PCR. Similar levels of abundance
were determined for each microRNA on both occasions, such that
the two abundance profiles (for all 32 microRNAs) were within less
than 1 CTdifference of one another (Figure 1E). Summarily, our
qRT-PCR based platform is sensitive, reproducible and specific,
capable of detecting accurately a few molecules of microRNA
present in a complex RNA background. Such sensitivity makes it
possible to use this platform to monitor the minute amount of
microRNA present in cell-free body fluids.
microRNAs are present in cell-free bodily fluids
When we initiated this study, there had been no demonstration
that microRNAs are present in cell-free body fluids. While this
manuscript was in preparation, it was reported that microRNAs
are detectable in serum . Here, a reliable protocol is presented
that we developed for extracting microRNAs from body fluids,
microRNA that is cell-free and DNA-free (see Materials and
Methods) and that can serve as the template in our qRT-PCR
To validate that microRNAs are indeed reproducibly detectable
in serum, RNA was extracted from the sera of two healthy
unrelated individuals and the levels of highly abundant micro-
RNAs examined (Figure 2A). We observed that microRNAs are
present at similar levels in both serum samples. This finding not
only establishes the reproducible presence of microRNAs in
serum, but also indicates that in general microRNA levels are
similar among individuals. Moreover, this finding supports our
premise that changes in the levels of specific microRNAs might
allow detection of clinical conditions. In parallel, using the same
methods we confirmed that microRNAs are also detectable in
other body fluids, such as urine, saliva, amniotic fluid and pleural
fluid (Figure 2B and data not shown). Of note, serum and urine
display different microRNA abundance profiles as might be
expected for two dissimilar biological fluids, further supporting our
hypothesis that bodily fluid microRNA profiles reflect physiology.
Serum MicroRNA as Biomarkers
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microRNAs in serum are sufficiently stable to serve as
In order for microRNAs in serum to be useful as clinical
biomarkers, they must be stable for reasonable periods of time,
and preferably during freeze-thaw cycles, to allow for routine
processing of clinical samples. Therefore, we investigated the
stability of serum microRNAs at room temperature and checked
the influence of freeze-thaw cycles on serum microRNA levels. We
found that the levels of different microRNAs in unfrozen serum do
not change substantially over a 4 hour period at room temper-
ature (Figure 3A). Moreover, serum microRNA levels are little
affected by twice freezing and re-thawing of serum samples
(Figure 3B). Thus, microRNAs in serum are sufficiently robust to
serve as practicable clinical biomarkers.
Figure 1. qRT-PCR can be used to monitor low microRNA levels specifically and sensitively. A) Schematic representation of the qRT-PCR
method. RNA is subjected to polyA polymerase reaction. Then, a universal RT reaction is performed that allows the amplification of all microRNAs as
well as mRNAs. The PCR amplification is performed using a reverse primer complementary to part of the oligodT primer and a forward primer, which
is homologous to a stretch in the microRNA sequence; in addition, the amplification reaction contains a TaqMan probe that covers part of the oligodT
primer sequence and some nucleotides complementary to the 39 sequence of the microRNA. B) Each synthetic RNA (hsa-let 7a, c & d) was subjected
to three independent qRT-PCR amplifications, where in each reaction there were present primers specific to only one of the three family members.
PCR amplification was observed only in the reaction where the primer matches the synthetic RNA. RNA amounts are described as percentages, each
relative to the level observed in the reaction containing primers matching the synthetic microRNA. ND is non-detectable. C) All three synthetic
microRNAs were mixed and subjected to qRT-PCR in the presence of the let-7d primer-probe set. In parallel, the same amount of synthetic let-7d as
used in the mixture was subjected alone to qRT-PCR. These parallel PCR reactions were repeated using reducing concentrations of let-7d synthetic
microRNA. At all tested concentrations of let-7d, it was amplified equivalently whether alone or in the presence of homologous family members. The
CTof let-7d is proportional to the input microRNA amount. D) Reducing concentrations (100-0.03%) of total RNA extracted from liver tissue (Ambion
Inc., No. 0360093B#) were mixed with total RNA extracted from brain tissue (Ambion Inc., No. 016P040305030A#) and the mixtures subjected to
qRT-PCR, where each reaction contained a primer specific to hsa-miR-122a. Hsa-miR-122a was detected linearly, even in samples where only 0.03%
liver RNA was spiked into brain RNA. When no liver RNA was introduced (100% brain RNA), hsa-miR-122a was not detected. E) The amounts of 32
different microRNAs in an RNA sample were examined on two independent occasions (by two different researchers) using the qRT-PCR platform
(microRNA level is represented as CTvalue). The two profiles were within less than 1 CTdifference of one another.
Serum MicroRNA as Biomarkers
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Serum microRNA profiles reflect physiological conditions
Finally, as a proof of concept, we investigated whether circulating
microRNAs can be used to identify clinical conditions. It has been
established that circulating maternal RNA contains placental
embryonic RNA . Therefore, we chose to compare the serum
microRNA abundance profiles of non pregnant versus pregnant
women, the latter in either their first or third trimester. We
measured the serum levels of 28 microRNAs, including microRNAs
reported to be placenta-specific [10,20] as well as broadly expressed
microRNAs. Box plots show relative microRNA levels in the sera of
10 non pregnant women, 10 women in the first trimester and 10
women in the third trimester (Figure 4A). The median fold changes
in microRNA levels comparing third trimester pregnant women to
non pregnant women are detailed in Table 1. MicroRNAs
expressed equivalently across all samples were used for normaliza-
tion. All of the placental microRNAs are found at higher levels in
the sera from pregnant women, their levels rising with gestational
age, and the levels of 12 microRNAs increased by more than 5-fold.
Figure 2. MicroRNAs are present in bodily fluids. A) microRNA levels in serum samples taken from 2 healthy individuals were measured. The
levels of 18 different microRNAs (blue circles, cycle thresholds (CT) values) and the 4 synthetic RNA ‘spike-ins’ (in the lower left part of the graph) were
found to be similar. B) To demonstrate that our extraction and evaluation methods can be applied to other body fluids, the same set of 20 microRNAs
examined in serum were assessed in urine samples from 2 healthy individuals. Some microRNAs were undetectable in the urine samples and
therefore are not shown. Notably, urine and serum samples demonstrate different microRNA abundance profiles.
Figure 3. MicroRNAs are stable during serum handling. A) microRNA stability in serum samples was monitored by extracting RNA from serum
samples kept for 1, 2 or 4 h at room temperature before freezing. The levels of 20 different microRNAs (blue circles), as well as of the 4 synthetic RNA
‘spike-ins’ (in the lower left part of the graph), were found to be similar across the 4 h time period. B) microRNA stability in serum samples was
monitored by extracting RNA from serum samples before and after freezing. The levels of 20 different microRNAs (blue circles), as well as of the 4
synthetic RNA ‘spike-ins’ (in the lower left part of the graph), were found to be similar following re-freezing and re-thawing of the sample.
Serum MicroRNA as Biomarkers
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Specifically, amounts of hsa-miR-526a and hsa-miR-527 are
dramatically higher in the sera of third trimester pregnant women
(elevated by more than 600 fold). Indeed, we found that the levels of
three placental microRNAs (hsa-miR-526a, hsa-miR-527 and hsa-
miR-520d-5p) could be used to accurately distinguish pregnant
from non pregnant women (Figure 5).
Figure 4. Differential amounts of four microRNAs in the sera of pregnant vs. non pregnant women. Box plots comparing microRNA
levels in the sera of 10 non pregnant women (A), 10 women in the first trimester (B), and 10 women in the third trimester (C). microRNA level is
specified as 50-CT, where CTis the cycle threshold of the PCR reaction. Results were normalized by subtracting the global microRNA level in the
sample (average CTof the 6 microRNAs chosen for normalization) from the level (CT) of each microRNA. A) The three placental microRNAs (miR-527,
miR-520d-5p and miR-526a) are highly abundant in the sera of pregnant women and their levels rise as pregnancy progresses. Hsa-let-7d levels are
also shown; this was one of the 6 microRNAs chosen for normalization as this microRNA exhibits similar abundance across the three groups. B)
microRNA miR-141 and miR-149 levels are mildly upregulated during pregnancy.
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While we were preparing these results for publication, another
research group reported that hsa-miR-141 and hsa-miR-149 are
present in serum and display increased abundance during
pregnancy . Therefore, we examined also the levels of these
two microRNAs in our serum samples. We found hsa-miR-141
and hsa-miR-149 levels to be higher by 4 and less than 2 fold,
respectively, in third trimester pregnant women relative to non
pregnant women (Table 1 and Figure 4B). These two microRNAs
are associated with epithelial tissues  that are not unique to
pregnancy and this may explain the small changes in their
amounts during pregnancy, which are insufficient to discriminate
pregnant from non pregnant women.
The results of this study clearly uphold our basic hypothesis that
microRNAs are present in bodily fluids and represent useful
clinical biomarkers. Importantly, we have developed reliable
methods for extracting microRNAs from bodily fluids and for
evaluating their abundance. Moreover, we have demonstrated that
microRNA serum levels reflect physiological conditions, such as
pregnancy, and can even be used to determine pregnancy stage.
We identified previously a large subset of microRNAs that are
expressed almost exclusively in the placenta [10,20] and found that a
of these placental microRNAs remains unclear, although it is
tempting to speculate that they contribute to specific morphological
placental features characteristic of primates and key to their evolution
. Future studies may reveal how these placental microRNAs
influence primate physiology. Here we show that many of these
microRNAs are found in maternal serum, at levels that increase with
gestational age. Easily accessible biomarkers for pregnancy compli-
cations and for various diseases are an urgent goal. In particular,
preeclampsia is a disorder affecting 5–8% of pregnancies that is a
leading global cause of maternal and infant illness or death [22,23].
The recently reported, distinctive expression of microRNAs in the
placenta in association with preeclampsia  highlights the
possibility that serum levels of particular microRNAs may serve as
future diagnostic biomarkers for preeclampsia.
This proof of concept study reveals the potential of body fluid
microRNAs to serve as practicable molecular markers for diverse
physiological and pathological conditions, especially those where
microRNAs have already been found to play a critical role, such as
cancer. Moreover, we demonstrate here the ease and reliability of
determining body fluid microRNA profiles and thus, pave the way
for their wide application, both in the research laboratory and in
Conceived and designed the experiments: SG EM ZB MH AC. Performed
the experiments: SG EM YY SB DL NY HC AC. Analyzed the data: SG
EM SB DL HB MK YG AC. Contributed reagents/materials/analysis
tools: NM. Wrote the paper: SG ZB MH AC.
Figure 5. ‘‘Pregnancy classification’’ according to the levels of
three microRNAs in the sera of pregnant vs. non pregnant
women. Discrimination of pregnant women from non pregnant
women based on microRNA levels in their sera. Blue circles represent
non pregnant women and red triangles represent pregnant women.
The location of each symbol in the plot represents the collective
expression of all three microRNAs in a given serum. The y axis indicates
the amount of mir-527, and the x axis indicates the average level of
miR-520d-5p and miR-526a.
Table 1. Serum microRNA levels - comparison between non
pregnant women and pregnant women in their third
microRNA delta CT
hsa-miR-515-5p9 511 6.90E-08
hsa-miR-520d-5p3.1 8.6 3.30E-07
hsa-miR-518d2.35 5.1 7.60E-03
hsa-miR-1412 4.0 3.90E-04
hsa-miR-519d 1.93.7 2.60E-02
hsa-miR-145 0.982.0 3.20E-02
For each microRNA, ‘‘delta CT’’ indicates the difference in median CTbetween
the serum of pregnant women in the third trimester (n=10) and non-pregnant
women (n=10). For each sample, the relative amount of the microRNAs was
normalized by subtracting the average CTof the non-placenta-specific
microRNAs. The fold change is the ratio of the median abundance in linear
space, equal to the exponent (base 2) of the delta CT. P-values are calculated by
a two-sided unpaired t-test.
Serum MicroRNA as Biomarkers
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