Circulating microRNAs as stable blood-based
markers for cancer detection
Patrick S. Mitchell†‡, Rachael K. Parkin†‡, Evan M. Kroh†‡, Brian R. Fritz†§, Stacia K. Wyman†,
Era L. Pogosova-Agadjanyan¶, Amelia Peterson†, Jennifer Noteboom?, Kathy C. O’Briant††, April Allen††,
Daniel W. Lin?††‡‡, Nicole Urban††, Charles W. Drescher††, Beatrice S. Knudsen††, Derek L. Stirewalt¶,
Robert Gentleman††, Robert L. Vessella?‡‡, Peter S. Nelson†¶, Daniel B. Martin†§§, and Muneesh Tewari†¶,¶¶
Divisions of†Human Biology,¶Clinical Research, and††Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109;
§§Institute for Systems Biology, Seattle, WA 98103;?Department of Urology, University of Washington, Seattle, WA 98195;
and‡‡Department of Veterans Affairs, Puget Sound Health Care System, Seattle, WA 98108
Communicated by Leland H. Hartwell, Fred Hutchinson Cancer Research Center, Seattle, WA, May 12, 2008 (received for review March 18, 2008)
Improved approaches for the detection of common epithelial
malignancies are urgently needed to reduce the worldwide mor-
bidity and mortality caused by cancer. MicroRNAs (miRNAs) are
small (?22 nt) regulatory RNAs that are frequently dysregulated in
cancer and have shown promise as tissue-based markers for cancer
classification and prognostication. We show here that miRNAs are
present in human plasma in a remarkably stable form that is
protected from endogenous RNase activity. miRNAs originating
from human prostate cancer xenografts enter the circulation, are
readily measured in plasma, and can robustly distinguish xe-
nografted mice from controls. This concept extends to cancer in
humans, where serum levels of miR-141 (a miRNA expressed in
prostate cancer) can distinguish patients with prostate cancer from
healthy controls. Our results establish the measurement of tumor-
derived miRNAs in serum or plasma as an important approach for
the blood-based detection of human cancer.
biomarker ? miR-141 ? plasma ? serum ? prostate cancer
reduce the worldwide health burden of cancer (1). Although
conventional strategies for blood-based biomarker discovery (e.g.,
using proteomic technologies) have shown promise, the develop-
ment of clinically validated cancer detection markers remains an
unmet challenge for many common human cancers (2). New
for cancer detection are urgently needed.
MicroRNAs (miRNAs) are small (typically ?22 nt in size)
specific mRNA targets and play important roles in a wide range of
physiologic and pathologic processes (3, 4). We hypothesized that
miRNAs could be an ideal class of blood-based biomarkers for
cancer detection because: (i) miRNA expression is frequently
dysregulated in cancer (5, 6), (ii) expression patterns of miRNAs in
human cancer appear to be tissue-specific (7), and (iii) miRNAs
have unusually high stability in formalin-fixed tissues (8–10). This
third point led us to speculate that miRNAs may have exceptional
stability in plasma and serum as well. We show here that miRNAs
are in fact present in clinical samples of plasma and serum in a
remarkably stable form. Furthermore, we establish proof-of-
principle for blood-based miRNA cancer detection by using both a
with prostate cancer. Our results lay the foundation for the devel-
opment of miRNAs as a novel class of blood-based cancer biomar-
kers and raise provocative questions regarding the mechanism of
stability and potential biological function of circulating miRNAs.
monitoring of common epithelial malignancies could greatly
Identification and Molecular Cloning of Endogenous miRNAs from
Human Plasma. PriorreportshavesuggestedthatRNAfromhuman
plasma (the noncellular component of blood remaining after
removing cells by centrifugation) is largely of low molecular weight
(11). We directly confirmed that human plasma contains small
size of total RNA isolated from plasma by using radioactive
labeling. PAGE and phosphorimaging of 5?32P-labeled plasma
RNA demonstrated RNA species ranging from 10 to 70 nt in size,
including a discernable species of size ?22 nt characteristic of most
miRNAs [supporting information (SI) Fig. S1]. The detected signal
was sensitive to RNase treatment but insensitive to DNase I
treatment, confirming that the signal originated from RNA
To directly determine whether miRNAs are present in human
plasma, we isolated the 18- to 24-nt RNA fraction from a human
plasma sample from a healthy donor (see SI Text for details on
blood collection and plasma RNA isolation) and used 5? and 3?
RNA–RNA linker ligations followed by RT-PCR amplification to
generate a small RNA cDNA library (Fig. 1A). Of the 125 clones
sequenced from this library, 27 corresponded to spiked-in size
marker oligos or linker–linker dimers. Ninety-one of the other 98
sequences (93%) corresponded to known miRNAs, providing
direct confirmation that mature miRNAs are present in human
plasma and indicating that the vast majority of 18- to 24-nt plasma
To quantitate specific miRNAs, we used TaqMan quantitative
RT-PCR (qRT-PCR) assays (12) to measure three miRNAs (miR-
15b, miR-16, and miR-24) in plasma from three healthy individuals.
These three miRNAs, chosen to represent moderate- to low-
abundance plasma miRNAs (based on the sequencing results
described above), were all readily detected in the plasma of each
133,970 copies/?l plasma, depending on the miRNA examined
Stability of Endogenous miRNAs in Human Plasma. Wenextsoughtto
investigate the stability of miRNAs in plasma, given that this is an
important prerequisite for utility as a biomarker. Incubation of
plasma at room temperature for up to 24 h (Fig. 2A Upper) or
had minimal effect on levels of miR-15b, miR-16, or miR-24 as
Author contributions: D.W.L., C.W.D., D.L.S., R.L.V., P.S.N., D.B.M., and M.T. designed
research; P.S.M., R.K.P., E.M.K., B.R.F., S.K.W., E.L.P.-A., A.P., J.N., K.C.O., and A.A. per-
formed research; N.U., B.S.K., D.L.S., and R.L.V. contributed new reagents/analytic tools;
B.R.F., and M.T. wrote the paper.
The authors declare no conflict of interest.
‡P.S.M., R.K.P, and E.M.K contributed equally to this work.
§Present address: Illumina, Inc., 9885 Towne Centre Drive, San Diego, CA 92121.
¶¶To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
July 29, 2008 ?
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measured by TaqMan qRT-PCR. Given that plasma has been
reported to contain high levels of RNase activity (13), we sought to
size or chemical structure, or whether it is caused by additional
extrinsic factors. We introduced synthetic miRNAs corresponding
to three known Caenorhabditis elegans miRNAs (cel-miR-39, cel-
miR-54, and cel-miR-238), chosen because of the absence of ho-
mologous sequences in humans, into human plasma either before
or after the addition of a denaturing solution that inhibits RNase
activity. RNA extraction followed by measurement of synthetic
added directly to plasma (the time between addition of synthetic
miRNAs and subsequent addition of denaturing solution was ?2
min), as compared with their addition after adding denaturing
solution to plasma (Fig. 2B). These results confirm the presence of
RNase activity in plasma and the sensitivity of naked miRNAs to
degradation. The levels of endogenous miRNAs (i.e., miR-15b,
miR-16, and miR-24) were not significantly altered in any of the
miRNAs exist in a form that is resistant to plasma RNase activity.
Comparison of miRNA Levels Between Plasma and Serum. Given that
blood is permitted to clot) are more plentiful than plasma samples
in many retrospective clinical sample repositories, we sought to
determine whether miRNA measurements are substantially differ-
miR-19b, and miR-24 in matched samples of serum or plasma
collected from a given individual at the same blood draw (Fig. 2C).
Measurements obtained from plasma or serum were strongly
correlated, indicating that both serum and plasma samples will be
suitable for investigations of miRNAs as blood-based biomarkers.
Tumor-Derived miRNAs Are Present in Plasma. Having demonstrated
that circulating miRNAs are detectable and stable in blood col-
lected from healthy individuals, we next sought to determine
whether tumor-derived miRNAs enter the circulation at levels
sufficient to be measurable as biomarkers for cancer detection. We
involves growth of the 22Rv1 human prostate cancer cell line in
NOD/SCID immunocompromised mice (14–17). We established a
cohort of 12 mice xenografted with 22Rv1 cells injected with
Matrigel and 12 control mice inoculated with Matrigel alone (Fig.
3A). Plasma was collected 28 days later (once tumors were well
established), and RNA was isolated for miRNA quantitation.
Because of the lack of an established endogenous miRNA control
plasma. (A) Cloning and sequencing of miRNAs
from human plasma. The schematic diagram
depicts the preparation of a small RNA library
from human plasma. Briefly, the 18- to 24-nt
a single donor (individual 006; described in Ta-
ble S6) was isolated by PAGE. Purified miRNAs
were then 3? and 5?ligated to single-stranded
oligonucleotides that contained universal
primer sequences for reverse transcription and
library of small RNA cDNA molecules that were
ligated into a plasmid vector (pCR4-TOPO) and
transformed into Escherichia coli. Inserts from a
total of 125 individual colonies yielded high-
a reference database of known miRNA se-
quences (miRBase Release v.10.1) (19) and to
GenBank. Seventy-three percent of sequences
corresponded to known miRNAs as shown. The
next most abundant species were matches to
the sequence of synthetic RNAs spiked in as
enously derived RNA sequences are considered,
miRNAs represent 93% (91 of 98) of the recov-
ered sequences. The miRNA read designated as
known let-7f miRNA except for a G-to-A substi-
tution at nucleotide position 15. (B) Quantifica-
plasma by TaqMan qRT-PCR. The graph indi-
cates the number of copies of each of three
representative miRNAs measured in plasma ob-
tained from three healthy individuals. In each
case, values represent the average of two repli-
cate reverse transcription reactions followed by
real-time PCR. For each miRNA assay, a dilution
to generate a standard curve that permitted
absolute quantification of molecules of
miRNA/?l plasma as shown here (see Fig. S2 for
standard curve plots). Values were median-
normalization controls spiked in immediately after addition of denaturing solution during RNA isolation (see SI Text for full details). The absence of amplification in
and negative controls are provided in Fig. S3).
Identification of miRNAs in human
www.pnas.org?cgi?doi?10.1073?pnas.0804549105Mitchell et al.
for plasma or serum, we introduced three synthetic C. elegans
miRNAs (described earlier) after the addition of denaturing solu-
to correct for technical variations in RNA recovery (detailed in SI
We first sought to establish that endogenous (murine) miRNAs
exist in mouse plasma and determine whether the presence of
cancer may lead to a general increase in plasma miRNAs, whether
they be tumor- or host-derived. miR-15b, miR-16, and miR-24
3B) and were not expressed at substantially different levels in
xenograft-bearing mice, indicating that the presence of tumor does
not lead to a generalized increase in plasma miRNAs.
miRNA qRT-PCR array (Applied Biosystems). We identified
two miRNAs, miR-629* and miR-660, that (i) were expressed in
not have known mouse homologs and therefore would be expected
to be tumor-specific markers in this setting (Table S1). We next
analyzed plasma samples from control and xenograft mice for the
levels of miR-629* and miR-660 by TaqMan qRT-PCR. Levels of
miR-629* and miR-660 were generally undetectable in the control
mice, whereas they were readily detected (ranging from 10 to 1,780
copies/?l plasma for miR-629* and 5,189–90,783 copies/?l for
miR-660) in all of the xenografted mice (Fig. 3C). Levels of both
miR-629* and miR-660 were able to independently differentiate
xenografted mice from controls with 100% sensitivity and 100%
specificity. These data establish proof of the principle that tumor-
derived miRNAs reach the circulation where their measurement in
plasma can serve as a means for cancer detection.
To understand the basis for the wide variation in miRNA
abundance observed among the different xenografted mice, we
in miRNA abundance across animals reflects, at least in part, the
differences in tumor burden.
Tumor-Derived miRNAs in Plasma Are Not Cell-Associated.Wesought
to further explore the mechanism of protection of tumor-derived
miRNAs from plasma RNase activity by testing the hypothesis that
they are present inside circulating tumor cells that might have
escaped pelleting during primary centrifugation for plasma isola-
experiment, we filtered pooled plasma generated from the xeno-
graft or control groups through a 0.22-?m filter, followed by RNA
extraction from the filtrate and the material retained on the filter
(referred to as the retentate). Measurement of miR-629* and
miR-660 by qRT-PCR in each of the samples demonstrated that
virtually all of the tumor-derived miRNAs passed through the
0.22-?m filter (Fig. S5A). As expected, tumor-derived miRNAs
were essentially undetectable in all of the samples from the control
group. In a second independent experiment, we subjected plasma
incubation or freeze-thawed multiple times. (Upper) The graphs show normalized Ct values for the indicated miRNAs measured in parallel aliquots of human
plasma samples incubated at room temperature for the indicated times. The experiment was carried out by using plasma from the two different individuals
amounts after initial plasma denaturation for RNA isolation (described in detail in SI Text). (Lower) The graphs show normalized Ct values for the indicated
miRNAs measured in parallel aliquots of human plasma samples subjected to the indicated number of cycles of freeze-thawing. Raw Ct values were normalized
across samples by using the same approach as described above. (B) Exogenously added miRNAs are rapidly degraded in plasma, whereas endogenous miRNAs
are stable. Three C. elegans miRNAs (chosen for the absence of sequence similarity to human miRNAs) were chemically synthesized and added either directly
to human plasma (from individual 003; described in Table S6) or added after the addition of denaturing solution (containing RNase inhibitors) to the plasma
(referred to as ‘‘denatured plasma’’). RNA was isolated from both plasma samples, and the abundance of each of the three C. elegans miRNAs was measured
by TaqMan qRT-PCR (Left), as was that of three endogenous plasma miRNAs (Right). Asterisks indicate that the abundance ratios of cel-miR-39, cel-miR-54, and
cel-miR-238 added to human plasma directly, relative to addition to denatured plasma, were 1.7 ? 10?5, 9.1 ? 10?6, and 1.1 ? 10?5, respectively and therefore
the average Ct values (average of two technical replicates) of the indicated miRNAs measured in serum and plasma samples collected from a given individual
at the same blood draw. Results from three different individuals are shown. miRNA measurements were highly correlated in both sample types. Results shown
for synthetic C. elegans miRNAs spiked into each plasma or serum sample (after addition of denaturing solution) demonstrate that experimental recovery of
miRNAs and robustness of subsequent qRT-PCR is not affected by whether it is plasma or serum that is collected.
Characterization of miRNA stability in human plasma. (A) miRNA levels remain stable when plasma is subjected to prolonged room temperature
Mitchell et al. PNAS ?
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pools from the xenograft and control groups to a series of two
remaining after the initial centrifugation used to collect plasma,
fragments). We assayed for miRNA expression in the starting
material, any pelleted material obtained from each centrifugation,
and the supernatant remaining after the 12,000 ? g centrifugation.
present in the supernatant of the 12,000 ? g spin. Taken together,
the data indicate that tumor-derived miRNAs are not associated
the possibility that circulating tumor cells or fragments of the same
may have been lysed during the process of blood collection or
plasma processing. Even if that is the case, however, our results
show that miRNAs that may have been released are ultimately
present in a stable, protected form of size much smaller than that
of a typical epithelial cell.
Detection of Human Prostate Cancer Based on Measurement of a
Prostate Cancer-Expressed miRNA in Serum. We next sought to
extend this approach to cancer detection in humans. We reasoned
that an ideal marker would be (i) expressed by the cancer cells at
moderate or high levels and (ii) present at very low or undetectable
levels in plasma from healthy individuals. We established a list of
likely blood-based miRNA biomarker candidates for prostate can-
cer by (i) compiling a list of miRNAs expressed in human prostate
cancer specimens based on published miRNA expression profiling
data (7, 18) and (ii) filtering out miRNAs detected in healthy
donor-derived plasma in our miRNA cloning experiment (Fig. 1A)
or detected on a microfluidic TaqMan qRT-PCR array analysis of
plasma from a normal healthy individual (details provided in SI
Text and see Table S8). This process generated a list of six leading
candidates (miR-100, miR-125b, miR-141, miR-143, miR-205, and
miR-296) for further investigation.
We chose to analyze these candidates in a case-control cohort of
serum samples collected from 25 individuals with metastatic pros-
tate cancer and 25 healthy age-matched male control individuals.
To efficiently screen multiple miRNA biomarker candidates, we
first generated two pools of serum aliquots derived from the
of the six candidate biomarker miRNAs by TaqMan qRT-PCR
assays. Results of this screen indicated that five of six of these
candidate miRNA biomarkers showed increased expression, al-
though to varying degrees, in the prostate cancer serum pool
compared with the healthy control group serum pool (Table S2).
For one of the candidates (miR-205), no conclusion could be
reached because miRNA levels in both pools were lower than the
limit of detection of the assay (as determined by a standard curve
of healthy control mice and their levels are not nonspecifically altered in cancer-bearing mice. Plasma levels of miR-15b, miR-16, and miR-24 were measured in
12 healthy control mice and 12 xenograft-bearing mice. The mature sequence of these miRNAs is perfectly conserved between mice and humans. Ct values were
converted to absolute number of copies/?l plasma by using a dilution series of known input quantities of synthetic target miRNA run on the same plate as the
spiked into plasma after denaturation for RNA isolation (details of the normalization method are provided in SI Text). (C) Tumor-derived miRNAs are detected
in plasma of xenograft-bearing mice and can distinguish cancer-bearing mice from controls. Plasma levels of miR-629* and miR-660 (two human miRNAs that
number of copies/?l plasma and normalized as described for B (see Table 5) threshold. Given that homologous miRNAs are not believed to exist in mice, the low
level of signal detected for a few mice in the control group, particularly for the miR-660 assay, is likely to represent nonspecific background amplification. As
These points are therefore not shown on the graph, even though plasma samples from the entire group of 12 mice in the control group were studied.
www.pnas.org?cgi?doi?10.1073?pnas.0804549105Mitchell et al.
Of all of the candidates, miR-141 showed the greatest differential
expression (46-fold overexpressed) in the prostate cancer pool
compared with the control pool (Table S2). We therefore focused
our study on miR-141 by measuring the abundance of this miRNA
in all of the individual serum samples comprising the case and
control groups. Consistent with results from the analysis of pooled
samples, serum levels of miR-141 were, in general, substantially
higher in cancer cases compared with controls (Fig. 4A). Compar-
ison of the two groups by a Wilcoxon two-sample test yielded W ?
63 with a P ? 1.47 ? 10?7, confirming a significant difference in
miR-141 levels between the two groups. Furthermore, serum levels
of miR-141 could detect individuals with cancer with 60% sensi-
a Receiver Operating Characteristic plot (Fig. 4B) reflects strong
separation between the two groups, with an area under the curve
(AUC) of 0.907. Comparison of miR-141 levels to prostate-specific
antigen (PSA) values among the prostate cancer patients demon-
strated Pearson and Spearman (rank) correlation coefficients of
?0.85 and ?0.62, indicating that miR-141 and PSA levels are
moderately correlated (Table S3). Serum levels of nonbiomarker
candidate miRNAs miR-16, miR-19b, and miR-24 were not signif-
icantly different between cases and controls, supporting the notion
that miR-141 is specifically elevated in prostate cancer, as opposed
to reflecting a nonspecific, generalized increase in serum miRNA
levels in the setting of cancer (Fig. 4C). Taken together, the results
extend to human cancer the concept that circulating miRNAs can
serve as markers for cancer detection.
miR-141 Is an Epithelial-Associated miRNA Expressed by Several
Common Human Cancers. miR-141 is a member of an evolution-
arily conserved family of miRNAs that includes, in humans,
miR-141, miR-200a, miR-200b, miR-200c, and miR-429 (19).
The expression of zebrafish homologs of this family, when
studied by in situ hybridization, was found to localize to various
epithelial tissues (20). To gain more insight into the potential
biological role of miR-141, we explored the large miRNA
expression profiling dataset generated by Lu et al. (7), who
profiled a diverse range of human cancer types. Consistent
with findings from the zebrafish studies, the expression of
miR-141 was tightly associated with expression in epithelial
samples compared with nonepithelial samples (Fig. S6), and
miR-141 was expressed in a wide range of common epithelial
cancers including breast, lung, colon, and prostate.
To determine the relative expression of this miR-141 specifically
both comparatively between the two cell types and relative to all
other known miRNAs within a cell type, we generated small RNA
libraries from primary cultures of human prostate epithelial and
stromal cells and subjected them to massively parallel sequencing
(detailed in SI Text). We found that miR-141 (and two of its family
members, miR-200b and miR-200c) was readily detected in the
prostate epithelial cell dataset but strikingly absent in the prostate
stromal cells (Table S4). In fact, of all miRNAs in this analysis,
epithelial cells relative to prostate stromal cells (Table S4). Taken
together, the data are consistent with the notion that miR-141 is an
epithelial-restricted miRNA that can be detected in the circulation
as a prostate cancer biomarker.
Our Results Establish That Tumor-Derived miRNAs, Detected in Plasma
or Serum, Can Serve as Circulating Biomarkers for Detection of a
Common Human Cancer Type. Although there is a long history of
investigation of circulating mRNA molecules as potential biomar-
kers (21), blood-based miRNA studies are in their infancy. Re-
cently, Chim et al. (22) reported the detection by qRT-PCR of
miRNAs of presumed placental origin in the plasma of pregnant
women, and Lawrie et al. (23) reported detecting elevations in
early reports, our study yielded (i) a more comprehensive view of
plasma miRNAs by direct cloning and sequencing from a plasma
small RNA library, (ii) unique results on miRNA stability that
provide a firm grounding for further investigation of this class of
tumor-derived miRNAs can enter the circulation even when orig-
inating from an epithelial cancer type (as compared with hemato-
poietic malignancies like lymphoma). Most importantly, our study
of miR-141 in prostate cancer patients demonstrates that serum
specificity and sensitivity, patients with cancer from healthy
associated miRNA miR-141. (A) Serum levels of miR-141 discriminate patients
with advanced prostate cancer from healthy controls. Serum levels of the
prostate cancer-expressed miRNA miR-141 were measured in 25 healthy con-
trol men and 25 patients with metastatic prostate cancer (clinical data on
of copies/?l serum by using a dilution series of known input quantities of
synthetic target miRNA run simultaneously (on the same plate) as the exper-
imental samples (dilution curves are provided in Fig. S2). Values shown have
been normalized by using measurements of C. elegans synthetic miRNA
controls spiked into plasma after denaturation for RNA isolation (details of
normalization method are provided in SI Text). The dashed line indicates a
100% specificity threshold. (B) Receiver Operating Characteristic (ROC) plot.
The data shown in A were used to draw the ROC plot shown. (C) Serum levels
of nontumor-associated miRNAs are not substantially different between pa-
tients with prostate cancer and controls. Serum levels of miR-16, miR-24, and
miR-19b were measured as negative controls as they are not expected to be
cancer-associated in the serum. Absolute quantification of miRNAs and data
normalization were carried out as described for A.
Detection of human prostate cancer by serum levels of tumor-
Mitchell et al. PNAS ?
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The available data indicate that miR-141 is expressed in an Download full-text
epithelial cell type-specific manner in a range of common human
cancers. Given this, we speculate that it could have value in the
setting of detecting cancer recurrence for cancer types for which
clinically validated blood biomarkers are lacking (e.g., lung cancer,
breast cancer, etc.). We also anticipate that advances in miRNA
qRT-PCR assay design and assay optimization, and the application
of alternative miRNA quantitation strategies, will substantially
improve the approach and will likely be needed to detect cancer at
blood-based miRNA markers that are specific for particular cancer
types will be discovered. The results presented here establish the
foundation and rationale to motivate future global investigations of
miRNAs as circulating cancer biomarkers for a variety of common
Our Study Raises Intriguing Questions Regarding the Mechanism of
miRNAs. The remarkable stability of miRNAs in clinical plasma
samples raises important and intriguing questions regarding the
mechanism by which miRNAs are protected from endogenous
inside exosomes that are secreted from cells. Exosomes are 50- to
90-nm (24), membrane-bound particles that have been reported to
be abundant in plasma (25) and that have recently been shown (in
cell culture studies) to contain miRNAs (26). Our results on
filtration (through a 0.22-?m filter) and differential centrifugation
of plasma containing tumor-derived miRNAs are certainly consis-
tent with this hypothesis (Fig. S5). Alternative explanations include
protection via association with other molecules (e.g., in a RNA–
protein complex) or modifications of the miRNAs that make them
resistant to RNase activity.
The high abundance of many circulating miRNAs also raises
provocative questions regarding their potential biological role as
extracellular messengers mediating short- and long-range cell–cell
communication, reminiscent of the similar role played by small
RNAs that spread within the organism in plants and C. elegans (27,
28). Additional studies will be needed to explore these exciting
Advantages and Potential of miRNAs as Blood-Based Cancer Biomar-
kers. The availability of powerful approaches for global miRNA
characterization and simple, universally applicable assays for quan-
titation (e.g., qRT-PCR) suggests that the discovery–validation
pipeline for miRNA biomarkers will be more efficient than tradi-
tional proteomic biomarker discovery–validation pipelines, which
typically encounter bottlenecks at the point of antibody generation
and quantitative assay development for validation of biomarker
candidates (2). In addition, the inherent regulatory function of
miRNAs makes it likely that many miRNAs expressed in tumor
tissue influence the biological behavior and clinical phenotype of
the tumor. As the functional roles of miRNAs in tumor biology are
unraveled, we envision that blood-based miRNA biomarkers that
predict clinical behavior and/or therapeutic response will be
Materials and Methods
Clinical Samples. Human plasma and serum samples from healthy donors or
patients with cancer were obtained with informed consent under institutional
Cancer Research Consortium, local healthy donors from the Seattle area, or the
Department of Urology, University of Washington. Details of sample collection
and processing and relevant corresponding clinical data are provided in SI Text.
RNA Isolation. Isolation of RNA from plasma or serum was carried out using a
modification of the mirVana PARIS kit (Ambion) that is described in detail in SI
s.c. injection of 7.5 ? 10522Rv1 cells per mouse. Mouse blood was collected by
cardiac puncture at 28 days after injection. Additional details of cell culture,
modifications as described in detail in SI Text.
Normalization of Experimental qRT-PCR Data from Plasma or Serum Using
Spiked-In C. elegans Controls. Three C. elegans miRNAs were chosen because of
a lack of sequence homology to human miRNAs and absence of empiric hybrid-
concentrations to be spiked-in were derived empirically to produce Ct values
blood from human plasma from normal healthy donors (Table S9). Synthetic
versions of the C. elegans miRNAs were spiked into plasma or serum after the
samples. Full details of the use of these controls to normalize qRT-PCR results
across independent RNA isolations are provided in SI Text.
Fred Hutchinson Cancer Research Center NOD/SCID core facility for technical
assistance with mouse xenograft experiments and F. Appelbaum, A. Geballe, J.
manuscript. This work was supported in part by Pacific Ovarian Cancer Research
(to R.L.V.), Pacific Northwest Prostate Cancer Specialized Program of Research
Excellence Grant P50 CA97186 (to P.S.N., M.T., and R.L.V.), Core Center of Excel-
lence in Hematology Pilot Grant P30 DK56465 (to M.T.), and the Paul Allen
Foundation for Medical Research.
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