|Genomic analysis of fetal nucleic acids in
Dennis Lo YM, Kwun Chiu RW.
Li Ka Shing Institute of Health Sciences and Department of Chemical
Pathology, Prince of Wales Hospital, The Chinese University of Hong
Kong, Shatin, New Territories, Hong Kong SAR, China
Annu Rev Genomics Hum Genet. 2012 Sep 22;13: 285-306
The 15 years since
the discovery of fetal DNA in maternal plasma have
witnessed remarkable developments in noninvasive prenatal diagnosis. An
understanding of biological parameters governing this phenomenon, such
as the concentration and molecular size of circulating fetal DNA, has
guided its diagnostic applications. Early efforts focused on the
detection of paternally inherited sequences, which were absent in the
maternal genome, in maternal plasma. Recent developments in precise
measurement technologies such as digital polymerase chain reaction
(PCR) have allowed the detection of minute allelic imbalances in plasma
and have catalyzed analysis of single-gene disorders such as the
hemoglobinopathies and hemophilia. The advent of massively parallel
sequencing has enabled the robust detection of fetal trisomies in
maternal plasma. Recent proof-of-concept studies have detected a
chromosomal translocation and a microdeletion and have deduced a
genome-wide genetic map of a fetus from maternal plasma. Understanding
the ethical, legal, and social aspects in light of such rapid
developments is thus a priority for future research.
|Nucleic acids in circulation: are they
harmful to the host?
Mittra I, Nair NK, Mishra PK.
Department of Translational Research, Advanced Centre for Treatment,
Research and Education in Cancer, Tata Memorial Centre, Kharghar, Navi
Mumbai 410 210, India
J Biosci. 2012 Jun;37(2): 301-312
It has been
estimated that 10(11) -10(12) cells, primarily of
haematogenous origin, die in the adult human body daily, and a similar
number is regenerated to maintain homeostasis. Despite the presence of
an efficient scavenging system for dead cells, considerable amounts of
fragmented genetic material enter the circulation in healthy
individuals. Elevated blood levels of extracellular nucleic acids have
been reported in various disease conditions; such as ageing and
age-related degenerative disorders, cancer; acute and chronic
inflammatory conditions, severe trauma and autoimmune disorders. In
addition to genomic DNA and nucleosomes, mitochondrial DNA is also
found in circulation, as are RNA and microRNA. There is extensive
literature that suggests that extraneously added nucleic acids have
biological actions. They can enter into cells in vitro and in vivo and
induce genetic transformation and cellular and chromosomal damage; and
experimentally added nucleic acids are capable of activating both
innate and adaptive immune systems and inducing a sterile inflammatory
response. The possibility as to whether circulating nucleic acids may,
likewise, have biological activities has not been explored. In this
review we raise the question as to whether circulating nucleic acids
may have damaging effects on the host and be implicated in ageing and
diverse acute and chronic human pathologies.
|Non-invasive prenatal diagnosis of
single-gene disorders from maternal blood
Bustamante-Aragonés A, Rodríguez de Alba M, Perlado S,
Trujillo-Tiebas MJ, Arranz JP, Díaz-Recasens J, Troyano-Luque J,
Gene. 2012 Aug 1;504(1): 144-149
(PD) is available for pregnancies at risk of
monogenic disorders. However, PD requires the use of invasive obstetric
techniques for fetal-sample collection and therefore, involves a risk
of fetal loss. Circulating fetal DNA in the maternal bloodstream is
being used to perform non-invasive prenatal diagnosis (NIPD). NIPD is a
challenging discipline because of the biological features of the
maternal blood sample. Maternal blood is an unequal mixture of small
(and fragmented) amounts of fetal DNA within a wide background of
maternal DNA. For this reason, initial NIPD studies have been based on
the analysis of specific paternally inherited fetal tracts not present
in the maternal genome so as to ensure their fetal origin. Following
this strategy, different NIPD studies have been carried out, such as
fetal-sex assessment for pregnancies at risk of X-linked disorders, RhD
determination, and analysis of single-gene disorders with a paternal
origin. The study of the paternal mutation can be used for fetal
diagnosis of dominant disorders or to more accurately assess the risk
of an affected child in case of recessive diseases. Huntington's
disease, cystic fibrosis, or achondroplasia are some examples of
diseases studied using NIPD. New technologies are opening NIPD to the
analysis of maternally inherited fetal tracts. NIPD of trisomy 21 is
the latest study derived from the use of next-generation sequencing
|Circulating micro-RNAs as potential
blood-based markers for early stage breast cancer detection
Schrauder MG, Strick R, Schulz-Wendtland R, Strissel PL, Kahmann L,
Loehberg CR, Lux MP, Jud SM, Hartmann A, Hein A, Bayer CM, Bani MR,
Richter S, Adamietz BR, Wenkel E, Rauh C, Beckmann MW, Fasching PA.
Department of Obstetrics and Gynaecology, University Hospital Erlangen,
Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany
PLoS One. 2012;7(1): e29770
MicroRNAs (miRNAs, miRs) are a class of small, non-coding
RNA molecules with relevance as regulators of gene expression thereby
affecting crucial processes in cancer development. MiRNAs offer great
potential as biomarkers for cancer detection due to their remarkable
stability in blood and their characteristic expression in many
different diseases. We investigated whether microarray-based miRNA
profiling on whole blood could discriminate between early stage breast
cancer patients and healthy controls.
performed microarray-based miRNA profiling on whole blood
of 48 early stage breast cancer patients at diagnosis along with 57
healthy individuals as controls. This was followed by a real-time
semi-quantitative Polymerase Chain Reaction (RT-qPCR) validation in a
separate cohort of 24 early stage breast cancer patients from a breast
cancer screening unit and 24 age matched controls using two
differentially expressed miRNAs (miR-202, miR-718).
RESULTS: Using the
significance level of p<0.05, we found that 59
miRNAs were differentially expressed in whole blood of early stage
breast cancer patients compared to healthy controls. 13 significantly
up-regulated miRNAs and 46 significantly down-regulated miRNAs in our
microarray panel of 1100 miRNAs and miRNA star sequences could be
detected. A set of 240 miRNAs that was evaluated by radial basis
function kernel support vector machines and 10-fold cross validation
yielded a specificity of 78.8%, and a sensitivity of 92.5%, as well as
an accuracy of 85.6%. Two miRNAs were validated by RT-qPCR in an
independent cohort. The relative fold changes of the RT-qPCR validation
were in line with the microarray data for both miRNAs, and
statistically significant differences in miRNA-expression were found
profiling in whole blood has potential as a novel
method for early stage breast cancer detection, but there are still
challenges that need to be addressed to establish these new biomarkers
in clinical use.
|Evaluation of circulating tumor cells and
circulating tumor DNA in non-small cell lung cancer: association with
clinical endpoints in a phase II clinical trial of pertuzumab and
Punnoose EA, Atwal S, Liu W, Raja R, Fine BM, Hughes BG, Hicks RJ,
Hampton GM, Amler LC, Pirzkall A, Lackner MR.
Department of Oncology Biomarker Development and Oncology Clinical
Development, Genentech, Inc, South San Francisco, California 94080, USA.
Clin Cancer Res. 2012 Apr 15;18(8): 2391-2401
levels or increases in circulating tumor cells (CTC)
portend poor prognosis in patients with epithelial cancers. Less is
known about CTCs as surrogate endpoints or their use for predictive
biomarker evaluation. This study investigated the utility of CTC
enumeration and characterization using the CellSearch platform, as well
as mutation detection in circulating tumor DNA (ctDNA), in patients
with advanced non-small cell lung cancer (NSCLC).
EXPERIMENTAL DESIGN: Forty-one patients were
enrolled in a single-arm
phase II clinical trial of erlotinib and pertuzumab. Peripheral blood
was analyzed for CTC enumeration, EGFR expression in CTCs, and
detection of oncogenic mutations in CTCs and ctDNA. Changes in CTC
levels were correlated with 2[18F]fluoro-2-deoxy-D-glucose-positron
emission tomographic (FDG-PET) and computed tomographic (CT) imaging
and survival endpoints.
RESULTS: CTCs were detected (≥ 1 CTC) at baseline in
78% of patients.
Greater sensitivity for mutation detection was observed in ctDNA than
in CTCs and detected mutations were strongly concordant with mutation
status in matched tumor. Higher baseline CTC counts were associated
with response to treatment by Response Evaluation Criteria in Solid
Tumors (RECIST, P = 0.009) and decreased CTC counts upon treatment were
associated with FDG-PET and RECIST response (P = 0.014 and P = 0.019)
and longer progression-free survival (P = 0.050).
CONCLUSION: These data provide evidence of a
decreases in CTC counts and radiographic response by either FDG-PET or
RECIST in patients with advanced NSCLC. These findings require
prospective validation but suggest a potential role for using CTC
decreases as an early indication of response to therapy and ctDNA for
real-time assessment of mutation status from blood.
|De novo sequencing of circulating miRNAs
identifies novel markers predicting clinical outcome of locally
advanced breast cancer
Wu X, Somlo G, Yu Y, Palomares MR, Li AX, Zhou W, Chow A, Yen Y, Rossi
JJ, Gao H, Wang J, Yuan YC, Frankel P, Li S, Ashing-Giwa KT, Sun G,
Wang Y, Smith R, Robinson K, Ren X, Wang SE.
Department of Cancer Biology, City of Hope Beckman Research Institute,
1500 Duarte Road, Duarte, CA 91010, USA.
J Transl Med. 2012 10:42.
MicroRNAs (miRNAs) have been recently detected in the
circulation of cancer patients, where they are associated with clinical
parameters. Discovery profiling of circulating small RNAs has not been
reported in breast cancer (BC), and was carried out in this study to
identify blood-based small RNA markers of BC clinical outcome.
METHODS: The pre-treatment sera of 42 stage II-III
locally advanced and
inflammatory BC patients who received neoadjuvant chemotherapy (NCT)
followed by surgical tumor resection were analyzed for marker
identification by deep sequencing all circulating small RNAs. An
independent validation cohort of 26 stage II-III BC patients was used
to assess the power of identified miRNA markers.
RESULTS: More than 800 miRNA species were detected
in the circulation,
and observed patterns showed association with histopathological
profiles of BC. Groups of circulating miRNAs differentially associated
with ER/PR/HER2 status and inflammatory BC were identified. The
relative levels of selected miRNAs measured by PCR showed consistency
with their abundance determined by deep sequencing. Two circulating
miRNAs, miR-375 and miR-122, exhibited strong correlations with
clinical outcomes, including NCT response and relapse with metastatic
disease. In the validation cohort, higher levels of circulating miR-122
specifically predicted metastatic recurrence in stage II-III BC
CONCLUSIONS: Our study indicates that certain miRNAs
can serve as
potential blood-based biomarkers for NCT response, and that miR-122
prevalence in the circulation predicts BC metastasis in early-stage
patients. These results may allow optimized chemotherapy treatments and
preventive anti-metastasis interventions in future clinical
|Genome-wide maps of circulating miRNA
biomarkers for ulcerative colitis
Duttagupta R, DiRienzo S, Jiang R, Bowers J, Gollub J, Kao J, Kearney
K, Rudolph D, Dawany NB, Showe MK, Stamato T, Getts RC, Jones KW.
Applied Reasearch Group, Affymetrix Inc, Santa Clara, California,
United States of America.
PLoS One. 2012;7(2): e31241
Disease--comprised of Crohn's Disease and Ulcerative
Colitis (UC)--is a complex, multi-factorial inflammatory disorder of
the gastrointestinal tract. In this study we have explored the utility
of naturally occurring circulating miRNAs as potential blood-based
biomarkers for non-invasive prediction of UC incidences. Whole genome
maps of circulating miRNAs in micro-vesicles, Peripheral Blood
Mononuclear Cells and platelets have been constructed from a cohort of
20 UC patients and 20 normal individuals. Through Significance Analysis
of Microarrays, a signature of 31 differentially expressed
platelet-derived miRNAs has been identified and biomarker performance
estimated through a non-probabilistic binary linear classification
using Support Vector Machines. Through this approach, classifier
measurements reveal a predictive score of 92.8% accuracy, 96.2%
specificity and 89.5% sensitivity in distinguishing UC patients from
normal individuals. Additionally, the platelet-derived biomarker
signature can be validated at 88% accuracy through qPCR assays, and a
majority of the miRNAs in this panel can be demonstrated to
sub-stratify into 4 highly correlated intensity based clusters.
Analysis of predicted targets of these biomarkers reveal an enrichment
of pathways associated with cytoskeleton assembly, transport, membrane
permeability and regulation of transcription factors engaged in a
variety of regulatory cascades that are consistent with a cell-mediated
immune response model of intestinal inflammation. Interestingly,
comparison of the miRNA biomarker panel and genetic loci implicated in
IBD through genome-wide association studies identifies a physical
linkage between hsa-miR-941 and a UC susceptibility loci located on Chr
20. Taken together, analysis of these expression maps outlines a
promising catalog of novel platelet-derived miRNA biomarkers of
clinical utility and provides insight into the potential biological
function of these candidates in disease pathogenesis.
|Circulating microRNAs in plasma of patients
with gastric cancers
Tsujiura M, Ichikawa D, Komatsu S, Shiozaki A, Takeshita H, Kosuga T,
Konishi H, Morimura R, Deguchi K, Fujiwara H, Okamoto K, Otsuji E.
Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural
University of Medicine, 465 Kajii-cho, Kawaramachihirokoji, Kamigyo-ku,
Kyoto 602-8566, Japan.
Br J Cancer. 2010 Mar 30;102(7): 1174-1179
examined plasma microRNA (miRNA) concentrations from
patients with gastric cancers (GCs) to assess their clinical
application for diagnosing and monitoring diseases.
METHODS: We initially investigated the
appropriateness of plasma miRNA
assay, and then compared plasma miRNA results with the expressions in
cancer tissues from eight GC patients, and also compared plasma miRNAs
between pre- and post-operative paired samples from 10 GC patients.
Then, plasma miRNAs (miR-17-5p, miR-21, miR-106a, miR-106b and let-7a)
were analysed in 69 GC patients and 30 healthy volunteers in total.
RESULTS: The initial analysis showed that miRNAs
were stable and
detectable in all plasma samples, and the plasma miRNA levels reflected
the tumour miRNAs in most cases. The levels of these miRNAs were
significantly reduced in post-operative samples. In large-scale
analysis, the plasma concentrations of miRNAs (miR-17-5p, miR-21,
miR-106a, miR-106b) were significantly higher in GC patients than
controls (P=0.05, 0.006, 0.008 and <0.001 respectively), whereas
let-7a was lower in GC patients (P=0.002). The values of the area under
the receiver-operating characteristic curve were 0.721 for the miR-106b
assay and 0.879 for the miR-106a/let-7a ratio assay.
CONCLUSION: Detection of circulating miRNAs might
complementary tumour markers for GC.
|Exosomes: Fit to deliver small RNA
Zomer A, Vendrig T, Hopmans ES, van Eijndhoven M, Middeldorp JM, Pegtel
Department of Pathology; Cancer Center Amsterdam; VU University Medical
Center; Amsterdam, The Netherlands.
Commun Integr Biol. 2010 Sep;3(5): 447-450
specialized membranous nano-sized vesicles derived from
endocytic compartments that are released by many cell types.
Microvesicles are distinctive from exosomes in that they are produced
by shedding of the plasmamembrane and usually larger in size (>1
µm). Exosome biogenesis involves the tightly controlled process
of inward budding from the limiting membrane of multivesicular bodies
(MVBs). This results in numerous intraluminal vesicles in the lumen of
MVBs that contain distinct protein repertoires. It has been suggested
that microvesicles shed by certain tumor cells hold functional
messenger RNA (mRNA) that may promote tumor progression. We discovered
that purified exosomes contain functional microRNAs (miRNAs) and small
RNA, but detected little mRNA. Although a clear and decisive
distinction between microvesicles and exosomes cannot be made and
different subsets of exosomes exist, we speculate that exosomes are
specialized in carrying small RNA including the class 22-25 nucleotide
regulatory miRNAs. To demonstrate this we developed a co-culture system
and found that exosomes are continuously secreted and transferred from
Epstein Barr virus (EBV)-infected cells to uninfected neighboring
cells. Throughout exosome transfer, the exogenous EBV-encoded miRNAs
were delivered to subcellular sites of miRNA-mediated gene repression.
Additionally, we found evidence that mature miRNAs are transferred
between circulating cells in humans, since we detected EBV-miRNAs in
non-infected cells in the peripheral blood of patients that include
monocytes and T cells. In this addendum we discuss these findings in
the context of recently published papers that advanced our current
knowledge of exosome physiology, (mi)RNA function and intercellular RNA
transfer. Based on this information we propose that an intercellular
(miRNA-based) mode of signal transmission may be well suited in
controlling space-confined processes such as the initiation of immune
responses in the secondary (peripheral) lymphoid tissues or in a tumor
microenvironment. Deciphering the molecular mechanism(s) that control
small RNA loading into exosomes and transfer to recipient cells in
vitro will provide new evidence for the physiological relevance of
vesicle-mediated intercellular communication in vivo.
|The majority of microRNAs detectable in
serum and saliva is concentrated in exosomes
Gallo A, Tandon M, Alevizos I, Illei GG.
Sjögren's Syndrome Clinic, Molecular Physiology and Therapeutics
Branch, National Institute of Dental and Craniofacial Research,
National Institutes of Health, Bethesda, Maryland, United States of
PLoS One. 2012;7(3):e30679
There is an
increasing interest in using microRNAs (miRNA) as
biomarkers in autoimmune diseases. They are easily accessible in many
body fluids but it is controversial if they are circulating freely or
are encapsulated in microvesicles, particularly exosomes. We
investigated if the majority of miRNas in serum and saliva are
free-circulating or concentrated in exosomes. Exosomes were isolated by
ultracentrifugation from fresh and frozen human serum and saliva. The
amount of selected miRNAs extracted from the exosomal pellet and the
exosome-depleted serum and saliva was compared by quantitative RT-PCR.
Some miRNAs tested are ubiquitously expressed, others were previously
reported as biomarkers. We included miRNAs previously reported to be
free circulating and some thought to be exosome specific. The purity of
exosome fraction was confirmed by electronmicroscopy and western blot.
The concentration of miRNAs was consistently higher in the exosome
pellet compared to the exosome-depleted supernatant. We obtained the
same results using an equal volume or equal amount of total RNA as
input of the RT-qPCR. The concentration of miRNA in whole,
unfractionated serum, was between the exosomal pellet and the
exosome-depleted supernatant. Selected miRNAs, which were detectable in
exosomes, were undetectable in whole serum and the exosome-depleted
supernantant. Exosome isolation improves the sensitivity of miRNA
amplification from human biologic fluids. Exosomal miRNA should be the
starting point for early biomarker studies to reduce the probability of
false negative results involving low abundance miRNAs that may be
missed by using unfractionated serum or saliva.
|Mechanism of transfer of functional
microRNAs between mouse dendritic cells via exosomes
Montecalvo A, Larregina AT, Shufesky WJ, Stolz DB, Sullivan ML,
Karlsson JM, Baty CJ, Gibson GA, Erdos G, Wang Z, Milosevic J, Tkacheva
OA, Divito SJ, Jordan R, Lyons-Weiler J, Watkins SC, Morelli AE.
Thomas E. Starzl Transplantation Institute, University of Pittsburgh
Medical Center, PA, USA.
Blood. 2012 Jan 19;119(3): 756-766
(DCs) are the most potent APCs. Whereas immature DCs
down-regulate T-cell responses to induce/maintain immunologic
tolerance, mature DCs promote immunity. To amplify their functions, DCs
communicate with neighboring DCs through soluble mediators,
cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles
(< 100 nm) derived from the endocytic pathway (termed exosomes)
represents a novel mechanism of DC-to-DC communication. The facts that
exosomes contain exosome-shuttle miRNAs and DC functions can be
regulated by exogenous miRNAs, suggest that DC-to-DC interactions could
be mediated through exosome-shuttle miRNAs, a hypothesis that remains
to be tested. Importantly, the mechanism of transfer of exosome-shuttle
miRNAs from the exosome lumen to the cytosol of target cells is
unknown. Here, we demonstrate that DCs release exosomes with different
miRNAs depending on the maturation of the DCs. By visualizing
spontaneous transfer of exosomes between DCs, we demonstrate that
exosomes fused with the target DCs, the latter followed by release of
the exosome content into the DC cytosol. Importantly, exosome-shuttle
miRNAs are functional, because they repress target mRNAs of acceptor
DCs. Our findings unveil a mechanism of transfer of exosome-shuttle
miRNAs between DCs and its role as a means of communication and
posttranscriptional regulation between DCs.
|Functional transfer of microRNA by exosomes
Utrecht University, the Netherlands
Blood. 2012 Jan 19;119(3): 646-648
Comment on -- Mechanism of transfer of functional
microRNAs between mouse dendritic cells via exosomes
|Secretory microRNAs as a versatile
Iguchi H, Kosaka N, Ochiya T.
Section for Studies on Metastasis; National Cancer Center Research
Institute; Chuo-ku, Tokyo Japan.
Commun Integr Biol. 2010 Sep;3(5): 478-481
role of microRNAs (miRNAs) is widely appreciated as a
fine-tuner to post-transcriptionally regulate the expression of
multiple genes in the cells of origin. Here, we highlight two
significant characteristics of miRNAs: (1) they are secreted from the
producing cells and (2) they can deliver the gene silencing signals
between living cells in vitro and in vivo. The circulation of miRNAs in
human body fluids can be provided with a logical explanation by our
discovery that the release of miRNAs is actively controlled through a
ceramide-dependent machinery associated with exosome secretion. This
finding can contribute to the development of circulating miRNAs as
diagnostic biomarkers for a variety of diseases. We also demonstrated
that secretory miR-16 was transferred into prostate cancer PC-3M cells
subcutaneously xenografted in nude mice, resulting in the suppression
of its target gene. This result suggests that faithfully to their
primary role, secretory miRNAs can function as a translational
inhibitor in recipient cells as well. In conclusion, miRNAs are
liberated from their incipient cells, whereby they can exert their full
potentials as a silence master of gene expressions.
|Presence and characterization of cell-free
seminal RNA in healthy individuals: implications for noninvasive
disease diagnosis and gene expression studies of the male reproductive
Huang S, Li H, Ding X, Xiong C.
Family Planning Research Institute, Tongji Medical College, Huazhong
University of Science and Technology, Wuhan 430030, China.
Clin Chem. 2009 Nov;55(11): 1967-1976
recently detected cell-free seminal RNA (cfsRNA) and set
out to study its concentration, integrity, stability in healthy
individuals, and mechanisms for its protection from ribonucleases.
METHODS: We quantified cfsRNA by
real-time PCR (RT-qPCR) targeting of the 5' region of the ACTB (actin,
beta) transcript. cfsRNA integrity was analyzed by microcapillary
electrophoresis and by amplification of full-length ACTB and DDX4 [DEAD
(Asp-Glu-Ala-Asp) box polypeptide 4] transcripts, including measurement
of the relative amounts of different regions of ACTB and DDX4
transcripts. Stability of cfsRNA was measured by time-course analysis
of different regions of ACTB and DDX4 transcripts. To investigate
whether cfsRNA was protected in complexed forms, we processed seminal
plasma in 2 ways: filtration through pores of different sizes and
Triton X-100 treatment before RNA recovery.
RESULTS: cfsRNA concentrations varied from 0.87-3.64
mg/L [mean (SD),
1.75 mg/L (0.92 mg/L)]. Most cfsRNA was present in partially degraded
forms, with smaller amounts of middle and 3' amplicons compared with 5'
amplicons. Although the 3' region of the DDX4 transcript was degraded
completely by 90 min, the 5' regions of ACTB and DDX4 transcripts were
stable up to 24 h. Filtration through 0.22-mum pores reduced ACTB and
DDX4 mRNA concentrations by 72% and 61%, respectively. Nearly all
seminal ACTB and DDX4 mRNA disappeared after Triton X-100 treatment.
CONCLUSIONS: Although cfsRNA was partially degraded,
diverse transcript species and was abundant, fairly stable, and
associated with particles in healthy individuals. cfsRNA may represent
a potential noninvasive biomarker of the male reproductive system and
of germline epigenetics.
|Identification of circulating miRNA
biomarkers based on global quantitative real-time PCR profiling
Kang K, Peng X, Luo J, Gou D.
College of Life Science, Shenzhen University, Shenzhen, Guangdong,
J Anim Sci Biotechnol. 2012 3(1): 4
MicroRNAs (miRNAs) are small noncoding RNAs (18-25
nucleotides) that regulate gene expression at the post-transcriptional
level. Recent studies have demonstrated the presence of miRNAs in the
blood circulation. Deregulation of miRNAs in serum or plasma has been
associated with many diseases including cancers and cardiovascular
diseases, suggesting the possible use of miRNAs as diagnostic
biomarkers. However, the detection of the small amount of miRNAs found
in serum or plasma requires a method with high sensitivity and
accuracy. Therefore, the current study describes polymerase chain
reaction (PCR)-based methods for measuring circulating miRNAs. Briefly,
the procedure involves four major steps: (1) sample collection and
preparation; (2) global miRNAs profiling using quantitative real-time
PCR (qRT-PCR); (3) data normalization and analysis; and (4) selection
and validation of miRNA biomarkers. In conclusion, qRT-PCR is a
promising method for profiling of circulating miRNAs as biomarkers.
quantitation of cell-free circulating microRNAs
Hastings ML, Palma J, Duelli DM.
Department of Cell Biology and Anatomy, Rosalind Franklin University of
Medicine and Science, North Chicago, IL, USA.
Methods. 2012 Aug 3. [Epub ahead of print]
microRNAs (miRNAs) that circulate in the blood are promising
surrogate biomarkers of disease and physiological processes. The ease
of quantifying specific miRNA species using made-to-order approaches
based on Taq-polymerase has led to numerous studies that have
identified changes in the abundance of circulating cell-free miRNA
species that correlate with pathology or other events. The growing
interest in developing miRNAs as blood biomarkers necessitates the
careful consideration of the unique properties of such body fluids that
can make the reproducible and quantitative assessment of RNA abundance
challenging. For example, enzymes involved in the amplification and
analysis of RNA can be affected by blood components that copurify with
miRNA. Thus, if miRNAs are to be effectively utilized as biomarkers, it
is important to establish standardized protocols for blood collection
and miRNA analysis to ensure accurate quantitation. Here we outline
several considerations, including the type of collection tube used in
sampling, the influence of added anticoagulants and stabilizers, sample
processing, enrichment of vesicular and other miRNA species, RNA
extraction approaches and enzyme selection, that affect quantitation of
miRNA isolated from plasma and should be considered in order to achieve
reproducible, sensitive and accurate quantitation.
|Changes in circulating microRNA levels
associated with prostate cancer
Bryant RJ, Pawlowski T, Catto JW, Marsden G, Vessella RL, Rhees B,
Kuslich C, Visakorpi T, Hamdy FC.
Nuffield Department of Surgical Sciences, University of Oxford, Oxford,
Br J Cancer. 2012 Feb 14;106(4): 768-774
aim of this study was to investigate the hypothesis
that changes in circulating microRNAs (miRs) represent potentially
useful biomarkers for the diagnosis, staging and prediction of outcome
in prostate cancer.
METHODS: Real-time polymerase chain reaction
analysis of 742 miRs was
performed using plasma-derived circulating microvesicles of 78 prostate
cancer patients and 28 normal control individuals to identify
differentially quantified miRs.
RESULTS: A total of 12 miRs were differentially
quantified in prostate
cancer patients compared with controls, including 9 in patients without
metastases. In all, 11 miRs were present in significantly greater
amounts in prostate cancer patients with metastases compared with those
without metastases. The association of miR-141 and miR-375 with
metastatic prostate cancer was confirmed using serum-derived exosomes
and microvesicles in a separate cohort of patients with recurrent or
non-recurrent disease following radical prostatectomy. An analysis of
five selected miRs in urine samples found that miR-107 and miR-574-3p
were quantified at significantly higher concentrations in the urine of
men with prostate cancer compared with controls.
CONCLUSION: These observations suggest that changes
concentration in prostate cancer patients may be identified by
analysing various body fluids. Moreover, circulating miRs may be used
to diagnose and stage prostate cancer.