Differential expression of microRNA in serum fractions and association of Argonaute 1 microRNAs with heart failure.

The serum or plasma microRNA (miRNA) molecules have been suggested as diagnostic and prognostic biomarkers, in various pathological conditions. However, these molecules are also found in different serum fractions, such as exosomes and Argonaute (Ago) protein complexes. Ago1 is the predominant Ago protein expressed in heart tissue. The objective of the study was to examine the hypothesis that Ago1-associated miRNAs may be more relevant to cardiac disease and heart failure compared with the serum. In total, 84 miRNA molecules were screened for their expression in the whole serum, exosomes and Ago1, and Ago2 complexes. Ago1-bound miR-222-3p, miR-497-5p and miR-21-5p were significantly higher, and let-7a-5p was significantly lower in HF patients compared with healthy controls, whereas no such difference was observed for those markers in the serum samples among the groups. A combination of these 4 miRNAs into an Ago1-HF score provided a ROC curve with an AUC of 1, demonstrating clear discrimination between heart failure patients and healthy individuals. Ago1 fraction might be a better and more specific platform for identifying HF-related miRNAs compared with the whole serum.

Identification of hundreds of conserved and nonconserved human microRNAs.

MicroRNAs are noncoding RNAs of approximately 22 nucleotides that suppress translation of target genes by binding to their mRNA and thus have a central role in gene regulation in health and disease. To date, 222 human microRNAs have been identified, 86 by random cloning and sequencing, 43 by computational approaches and the rest as putative microRNAs homologous to microRNAs in other species. To prove our hypothesis that the total number of microRNAs may be much larger and that several have emerged only in primates, we developed an integrative approach combining bioinformatic predictions with microarray analysis and sequence-directed cloning. Here we report the use of this approach to clone and sequence 89 new human microRNAs (nearly doubling the current number of sequenced human microRNAs), 53 of which are not conserved beyond primates. These findings suggest that the total number of human microRNAs is at least 800.

Novel microRNA-based assay demonstrates 92% agreement with diagnosis based on clinicopathologic and management data in a cohort of patients with carcinoma of unknown primary.

BACKGROUND: Cancer of unknown or uncertain primary is a major diagnostic and clinical challenge, since identifying the tissue-of-origin of metastases is crucial for selecting optimal treatment. MicroRNAs are a family of non-coding, regulatory RNA molecules that are tissue-specific, with a great potential to be excellent biomarkers. METHODS: In this study we tested the performance of a microRNA-based assay in formalin-fixed paraffin-embedded samples from 84 CUP patients. RESULTS: The microRNA based assay agreed with the clinical diagnosis at presentation in 70% of patients; it agreed with the clinical diagnosis obtained after patient management, taking into account response and outcome data, in 89% of patients; it agreed with the final clinical diagnosis reached with supplemental immunohistochemical stains in 92% of patients, indicating a 22% improvement in agreement from diagnosis at presentation to the final clinical diagnosis. In 18 patients the assay disagreed with the presentation diagnosis and was in agreement with the final clinical diagnosis, which may have resulted in the administration of more effective chemotherapy. In three out of four discordant cases in which supplemental IHC was performed, the IHC results validated the assay's molecular diagnosis. CONCLUSIONS: This novel microRNA-based assay shows high accuracy in identifying the final clinical diagnosis in a real life CUP patient cohort and could be a useful tool to facilitate administration of optimal therapy.

Predicting progression of bladder urothelial carcinoma using microRNA expression.

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Recurrence and progression prediction in urothelial cancer is currently based on clinical and pathological factors: tumour grade, tumour stage, number of lesions, tumour size, previous recurrence rate, and presence of concomitant carcinoma in situ. These factors are not specific enough to predict progression and ∼50% of patients diagnosed as high risk in fact do not progress within 3 years. Patient follow-up is both expensive and unpleasant (frequent invasive cystoscopies). Molecular biomarkers, including microRNAs have been studied to provide additional prognostic information for these patients, but to date no molecular biomarker has become the 'gold standard' for patient diagnosis and follow-up. We used Rosetta Genomics' highly specific microRNA expression profiling platforms to study the prognostic role of microRNAs in bladder cancer. Using microdissection we chose specific tumour microRNAs to study in order to avoid background contamination. Tumour progression was associated with altered levels of microRNAs. In particular, high expression levels of miR-29c* were associated with a good prognosis. The study found that the use of microRNAs for determining progression and invasiveness for patients with urothelial cancer could potentially have a substantial impact on the treatment and follow-up individual patients. OBJECTIVE: To identify microRNAs that could be useful as prognostic markers for non-muscle-invasive (NMI) bladder carcinoma. PATIENTS AND METHODS: Formalin-fixed, paraffin-embedded samples of 108 NMI bladder carcinomas, and 29 carcinomas invading bladder muscle were collected, and microRNA expression levels were measured using microarrays. For 19 samples, microdissection was performed to compare microRNA expression between the tumour and surrounding tissue. MicroRNAs that were found to be unrelated to the tumour itself were excluded as potential prognostic markers. RESULTS: Expression profiles identified microRNAs that were differentially expressed in NMI tumours from patients who later progressed to carcinoma invading bladder muscle compared with NMI tumours from patients that did not progress. The microRNA profile of tumours invading the bladder muscle was more similar to that of NMI tumours from patients who later progressed, than to that of the same-stage NMI tumours from patients who did not later progress. The expression level of one microRNA, miR-29c*, was significantly under-expressed in tumours that progressed and could be used to stratify patients with T1 disease into risk groups. CONCLUSIONS: MicroRNAs can be useful biomarkers for prognosis in patients with urothelial carcinoma. In our study, expression levels of several microRNAs, including miR-29c* identified high- and low-risk groups. These biomarkers show promise for the stratification of patients with bladder cancer.

Finding quasi-modules of human and viral miRNAs: a case study of human cytomegalovirus (HCMV).

BACKGROUND: MicroRNAs (miRNAs) are important regulators of gene expression encoded by a variety of organisms, including viruses. Although the function of most of the viral miRNAs is currently unknown, there is evidence that both viral and host miRNAs contribute to the interactions between viruses and their hosts. miRNAs constitute a complex combinatorial network, where one miRNA may target many genes and one gene may be targeted by multiple miRNAs. In particular, viral and host miRNAs may also have mutual target genes. Based on published evidence linking viral and host miRNAs there are three modes of mutual regulation: competing, cooperating, and compensating modes. RESULTS: In this paper we explore the compensating mode of mutual regulation upon Human Cytomegalovirus (HCMV) infection, when host miRNAs are down regulated and viral miRNAs compensate by mimicking their function. To achieve this, we develop a new algorithm which finds groups, called quasi-modules, of viral and host miRNAs and their mutual target genes, and use a new host miRNA expression data for HCMV-infected and uninfected cells. For two of the reported quasi-modules, supporting evidence from biological and medical literature is provided. CONCLUSIONS: The modules found by our method may advance the understanding of the role of miRNAs in host-viral interactions, and the genes in these modules may serve as candidates for further experimental validation.

A second-generation microRNA-based assay for diagnosing tumor tissue origin.

BACKGROUND: Cancers of unknown primary origin (CUP) constitute 3%-5% (50,000 to 70,000 cases) of all newly diagnosed cancers per year in the United States. Including cancers of uncertain primary origin, the total number increases to 12%-15% (180,000 to 220,000 cases) of all newly diagnosed cancers per year in the United States. Cancers of unknown/uncertain primary origins present major diagnostic and clinical challenges because the tumor tissue of origin is crucial for selecting optimal treatment. MicroRNAs are a family of noncoding, regulatory RNA genes involved in carcinogenesis. MicroRNAs that are highly stable in clinical samples and tissue specific serve as ideal biomarkers for cancer diagnosis. Our first-generation assay identified the tumor of origin based on 48 microRNAs measured on a quantitative real-time polymerase chain reaction platform and differentiated 25 tumor types. METHODS: We present here the development and validation of a second-generation assay that identifies 42 tumor types using a custom microarray. A combination of a binary decision-tree and a k-nearest-neighbor classifier was developed to identify the tumor of origin based on the expression of 64 microRNAs. RESULTS: Overall assay sensitivity (positive agreement), measured blindly on a validation set of 509 independent samples, was 85%. The sensitivity reached 90% for cases in which the assay reported a single answer (>80% of cases). A clinical validation study on 52 true CUP patients showed 88% concordance with the clinicopathological evaluation of the patients. CONCLUSION: The abilities of the assay to identify 42 tumor types with high accuracy and to maintain the same performance in samples from patients clinically diagnosed with CUP promise improved utility in the diagnosis of cancers of unknown/uncertain primary origins.

Vaccinia virus infection suppresses the cell microRNA machinery.

MicroRNAs are key players in the regulation of gene expression by posttranscriptional suppression. They are involved in physiological processes, and thus their deregulation may contribute to the development of diseases and progression of cancer. Virus-encoded microRNAs and microRNAs of host origin play an important role in controlling the virus life cycle and immunity. The aim of this study was to determine the effect of vaccinia virus (VACV) infection on the expression of host-encoded microRNAs. A marked general suppression of most microRNAs in the infected cells was observed within 24 hours after VACV infection of a number of cell types. We demonstrate that this suppression was associated with abrogation of expression of the Dicer1 enzyme, which is a key enzyme in the generation of microRNAs.

Discovery of microRNAs and other small RNAs in solid tumors.

MicroRNAs (miRNAs) are ∼22-nt long, non-coding RNAs that regulate gene silencing. It is known that many human miRNAs are deregulated in numerous types of tumors. Here we report the sequencing of small RNAs (17-25 nt) from 23 breast, bladder, colon and lung tumor samples using high throughput sequencing. We identified 49 novel miRNA and miR-sized small RNAs. We further validated the expression of 10 novel small RNAs in 31 different types of blood, normal and tumor tissue samples using two independent platforms, namely microarray and RT-PCR. Some of the novel sequences show a large difference in expression between tumor and tumor-adjacent tissues, between different tumor stages, or between different tumor types. We also report the identification of novel small RNA classes in human: highly expressed small RNA derived from Y-RNA and endogenous siRNA. Finally, we identified dozens of new miRNA sequence variants that demonstrate the existence of miRNA-related SNP or post-transcriptional modifications. Our work extends the current knowledge of the tumor small RNA transcriptome and provides novel candidates for molecular biomarkers and drug targets.

HIV-1 TAR miRNA protects against apoptosis by altering cellular gene expression.

BACKGROUND: RNA interference is a gene regulatory mechanism that employs small RNA molecules such as microRNA. Previous work has shown that HIV-1 produces TAR viral microRNA. Here we describe the effects of the HIV-1 TAR derived microRNA on cellular gene expression. RESULTS: Using a variation of standard techniques we have cloned and sequenced both the 5' and 3' arms of the TAR miRNA. We show that expression of the TAR microRNA protects infected cells from apoptosis and acts by down-regulating cellular genes involved in apoptosis. Specifically, the microRNA down-regulates ERCC1 and IER3, protecting the cell from apoptosis. Comparison to our cloned sequence reveals possible target sites for the TAR miRNA as well. CONCLUSION: The TAR microRNA is expressed in all stages of the viral life cycle, can be detected in latently infected cells, and represents a mechanism wherein the virus extends the life of the infected cell for the purpose of increasing viral replication.

Multicentre validation of a microRNA-based assay for diagnosing indeterminate thyroid nodules utilising fine needle aspirate smears.

AIMS: The distinction between benign and malignant thyroid nodules has important therapeutic implications. Our objective was to develop an assay that could classify indeterminate thyroid nodules as benign or suspicious, using routinely prepared fine needle aspirate (FNA) cytology smears. METHODS: A training set of 375 FNA smears was used to develop the microRNA-based assay, which was validated using a blinded, multicentre, retrospective cohort of 201 smears. Final diagnosis of the validation samples was determined based on corresponding surgical specimens, reviewed by the contributing institute pathologist and two independent pathologists. Validation samples were from adult patients (≥18 years) with nodule size >0.5 cm, and a final diagnosis confirmed by at least one of the two blinded, independent pathologists. The developed assay, RosettaGX Reveal, differentiates benign from malignant thyroid nodules, using quantitative RT-PCR. RESULTS: Test performance on the 189 samples that passed quality control: negative predictive value: 91% (95% CI 84% to 96%); sensitivity: 85% (CI 74% to 93%); specificity: 72% (CI 63% to 79%). Performance for cases in which all three reviewing pathologists were in agreement regarding the final diagnosis (n=150): negative predictive value: 99% (CI 94% to 100%); sensitivity: 98% (CI 87% to 100%); specificity: 78% (CI 69% to 85%). CONCLUSIONS: A novel assay utilising microRNA expression in cytology smears was developed. The assay distinguishes benign from malignant thyroid nodules using a single FNA stained smear, and does not require fresh tissue or special collection and shipment conditions. This assay offers a valuable tool for the preoperative classification of thyroid samples with indeterminate cytology.

Analytical validity of a microRNA-based assay for diagnosing indeterminate thyroid FNA smears from routinely prepared cytology slides.

BACKGROUND: The majority of thyroid nodules are diagnosed using fine-needle aspiration (FNA) biopsies. The authors recently described the clinical validation of a molecular microRNA-based assay, RosettaGX Reveal, which can diagnose thyroid nodules as benign or suspicious using a single stained FNA smear. This paper describes the analytical validation of the assay. METHODS: More than 800 FNA slides were tested, including slides stained with Romanowsky-type and Papanicolaou stains. The assay was examined for the following features: intranodule concordance, effect of stain type, minimal acceptable RNA amounts, performance on low numbers of thyroid cells, effect of time since sampling, and analytical sensitivity, specificity, and reproducibility. RESULTS: The assay can be run on FNA slides for which as little as 1% of the cells are thyroid epithelial cells or from which only 5 ng of RNA have been extracted. Samples composed entirely of blood failed quality control and were not classified. Stain type did not affect performance. All slides were stored at room temperature. However, the length of time between FNA sampling and processing did not affect assay performance. There was a high level of concordance between laboratories (96%), and the concordance for slides created from the same FNA pass was 93%. CONCLUSIONS: The microRNA-based assay was robust to various physical processing conditions and to differing sample characteristics. Given the assay's performance, robustness, and use of routinely prepared FNA slides, it has the potential to provide valuable aid for physicians in the diagnosis of thyroid nodules. Cancer Cytopathol 2016;124:711-21. © 2016 Rosetta Genomics. Cancer Cytopathology published by Wiley Periodicals, Inc. on behalf of American Cancer Society.

Specific MicroRNAs Differentiate Adrenocortical Adenomas from Carcinomas and Correlate With Weiss Histopathologic System.

MicroRNAs (miRs) play a central role in regulating gene expression and are strongly associated with cancer development. This study sought to determine if adrenocortical carcinomas can be differentiated from adenomas by their miR profiles and to correlate the findings with the histologic Weiss system for identifying malignancy in adrenocortical tumors (ACTs). Forty-six primary and 2 recurrent ACTs retrieved from the files of the pathology department of a tertiary medical center were evaluated blindly for the Weiss criteria. High-quality RNA was extracted, and miR expression was evaluated with microarrays and quantitative reverse-transcriptase polymerase chain reaction. The Weiss system defined 17 tumors as carcinomas and 29 as adenomas. On microarray analysis, over a dozen miRs were upregulated or downregulated in carcinomas compared with adenomas. Upregulation of miR-503 was the best single discriminator of malignancy. The combination of miR-34a and miR-497 underexpression discriminated carcinomas from adenomas with 100% sensitivity and 96% specificity. Statistical analysis revealed a high level of correspondence between the Weiss system and miR expression. In conclusion, miR expression can accurately identify malignant ACTs with equal efficiency to the Weiss system. miR analysis may have added value in tumors with borderline features that are difficult to interpret histopathologically.

Relation of reduced expression of MiR-150 in platelets to atrial fibrillation in patients with chronic systolic heart failure.

Atrial fibrillation (AF) is associated with poor prognosis in patients with heart failure (HF). Although platelets play an important role in rendering a prothrombotic state in AF, the exact mechanism by which the effect is mediated is still debated. MicroRNAs (miRNAs), which have been shown to be involved in a variety of cardiovascular conditions, are abundant in platelets and in a cell-free form in the circulation. In the present study, we performed a genome-wide screen for miRNA expression in platelets of patients with systolic HF and in controls without cardiac disease, in pursuit of specific miRNAs that are associated with the presence of AF. MiRNA expression was measured in platelets from 50 patients with systolic HF and 50 controls, of which, samples from 41 patients with HF and 35 controls were used in the final analysis because of a quality control process. MiR-150 expression was 3.2-fold lower (p = 0.0003) in platelets of patients with HF with AF relative to those without AF. A similar effect was seen in serum samples from the same patients, in which miR-150 levels were 1.5-fold lower (p = 0.004) in patients with HF with AF. Furthermore, the serum levels of miR-150 were correlated to platelet levels in patients with AF (r = 0.65, p = 0.0087). In conclusion, miR-150 expression levels in platelets of patients with systolic HF with AF are significantly reduced and correlated to the cell-free circulating levels of this miRNA.

Development and validation of a microRNA-based diagnostic assay for classification of renal cell carcinomas.

Renal cancers account for more than 3% of adult malignancies and cause more than 13,000 deaths per year in the US alone. The four most common types of kidney tumors include the malignant renal cell carcinomas; clear cell, papillary, chromophobe and the benign oncocytoma. These histological subtypes vary in their clinical course and prognosis, and different clinical strategies have been developed for their management. In some kidney tumor cases it can be very difficult for the pathologist to distinguish between tumor types on the basis of morphology and immunohistochemistry (IHC). In this publication we present the development and validation of a microRNA-based assay for classifying primary kidney tumors. The assay, which classifies the four main kidney tumor types, was developed based on the expression of a set of 24 microRNAs. A validation set of 201 independent samples was classified using the assay and analyzed blindly. The assay produced results for 92% of the samples with an accuracy of 95%.

Global microRNA profiling in favorable prognosis subgroups of cancer of unknown primary (CUP) demonstrates no significant expression differences with metastases of matched known primary tumors.

No data exist on biologic differences between Cancer of unknown primary (CUP) and metastatic solid tumors of known primary site. We assigned a primary tissue of origin in 40 favorable CUP patients (A: serous peritoneal carcinomatosis n = 14, B: axillary adenocarcinoma n = 8, C: upper squamous cervical adenopathy n = 18) by means of a 64-microRNA assay. Subsequently, we profiled the expression of 733 microRNAs (miRs) in the CUP cases and compared results with metastases from 20 ovarian carcinomas, 10 breast adenocarcinomas, 20 squamous head neck or lung tumors. In the Peritoneal CUP versus Ovarian (Known Primary Metastases) KPM comparison, a total of 12 miR were significantly differentially expressed: higher than twofold expression difference in CUP was seen only for miR-513a-5p (3.7-fold upregulated) and miR-483-5p (2.5-fold upregulated), while miR-708 exhibited a twofold downregulation. In the Breast CUP versus Breast KPM comparison, only miR-29c that were downregulated in CUP by 2.7-fold satisfied the FDR threshold. miR-30e and miR-27b, downregulated in ovarian CUPs versus KPMs, were also non-significantly downregulated in breast CUP by 2.0- and 1.4-fold respectively. Six miRs, which belong to the 17-92 oncocluster showed a trend of upregulation in Breast CUP versus Breast KPM cases. A CUP signature remains elusive.

Differential diagnosis of hepatocellular carcinoma from metastatic tumors in the liver using microRNA expression.

Distinguishing hepatocellular carcinoma from metastatic tumors in the liver is of great practical importance, with significant therapeutic and prognostic implications. This differential diagnosis can be difficult because metastatic cancers in the liver, especially adenocarcinomas, may mimic the morphology and immunoexpression of hepatocellular carcinoma. Biomarkers that are specifically expressed in either hepatocellular carcinoma or metastatic adenocarcinoma can therefore be useful diagnostic tools. To find such biomarkers, we studied microRNA expression in 144 tumor samples using custom microarrays. Hsa-miR-141 and hsa-miR-200c, microRNAs that promote epithelial phenotypes, had significantly higher levels in non-hepatic epithelial tumors. In contrast, endothelial-associated hsa-miR-126 showed higher expression levels in hepatocellular carcinomas. Combinations of these microRNAs accurately identified primary hepatocellular carcinoma from metastatic adenocarcinoma in the liver. These findings were validated using quantitative real-time PCR to measure microRNA expression in additional samples. Thus, the tissue-specific expression patterns of microRNAs make them useful biomarkers for the diagnosis of liver malignancies.

A diagnostic assay based on microRNA expression accurately identifies malignant pleural mesothelioma.

The definitive identification of malignant pleural mesothelioma (MPM) has significant clinical implications, yet other malignancies often involve the lung pleura, confounding the diagnosis of MPM. In the absence of accurate markers, MPM can be difficult to distinguish from peripheral lung adenocarcinoma and metastatic epithelial cancers. MicroRNA expression is tissue-specific and highly informative for identifying tumor origin. We identified microRNA biomarkers for the differential diagnosis of MPM and developed a standardized microRNA-based assay. Formalin-fixed, paraffin-embedded samples of 33 MPM and 210 carcinomas were used for assay development. Using microarrays, we identified microRNAs differentially expressed between MPM and various carcinomas. Hsa-miR-193-3p was overexpressed in MPM, while hsa-miR-200c and hsa-miR-192 were overexpressed in peripheral lung adenocarcinoma and carcinomas that frequently metastasize to lung pleura. We developed a standardized diagnostic assay based on the expression of these microRNAs. The assay reached a sensitivity of 100% and a specificity of 94% in a blinded validation set of 68 samples from the lung and pleura. This diagnostic assay can provide a useful tool in the differential diagnosis of MPM from other malignancies in the pleura.

Comprehensive gene and microRNA expression profiling reveals a role for microRNAs in human liver development.

BACKGROUND AND AIMS: microRNAs (miRNAs) are small noncoding RNAs that regulate cognate mRNAs post-transcriptionally. miRNAs have been implicated in regulating gene expression in embryonic developmental processes, including proliferation and differentiation. The liver is a multifunctional organ, which undergoes rapid changes during the developmental period and relies on tightly-regulated gene expression. Little is known regarding the complex expression patterns of both mRNAs and miRNAs during the early stages of human liver development, and the role of miRNAs in the regulation of this process has not been studied. The aim of this work was to study the impact of miRNAs on gene expression during early human liver development. METHODS: Global gene and miRNA expression were profiled in adult and in 9-12w human embryonic livers, using high-density microarrays and quantitative RT-PCR. RESULTS: Embryonic liver samples exhibited a gene expression profile that differentiated upon progression in the developmental process, and revealed multiple regulated genes. miRNA expression profiling revealed four major expression patterns that correlated with the known function of regulated miRNAs. Comparison of the expression of the most regulated miRNAs to that of their putative targets using a novel algorithm revealed a significant anti-correlation for several miRNAs, and identified the most active miRNAs in embryonic and in adult liver. Furthermore, our algorithm facilitated the identification of TGFbeta-R1 as a novel target gene of let-7. CONCLUSIONS: Our results uncover multiple regulated miRNAs and genes throughout human liver development, and our algorithm assists in identification of novel miRNA targets with potential roles in liver development.

MiR-92b and miR-9/9* are specifically expressed in brain primary tumors and can be used to differentiate primary from metastatic brain tumors.

A recurring challenge for brain pathologists is to diagnose whether a brain malignancy is a primary tumor or a metastasis from some other tissue. The accurate diagnosis of brain malignancies is essential for selection of proper treatment. MicroRNAs are a class of small non-coding RNA species that regulate gene expression; many exhibit tissue-specific expression and are misregulated in cancer. Using microRNA expression profiling, we found that hsa-miR-92b and hsa-miR-9/hsa-miR-9* are over-expressed, specifically in brain primary tumors, as compared to primary tumors from other tissues and their metastases to the brain. By considering the expression of only these two microRNAs, it is possible to distinguish between primary and metastatic brain tumors with very high accuracy. These microRNAs thus represent excellent biomarkers for brain primary tumors. Previous reports have found that hsa-miR-92b and hsa-miR-9/hsa-miR-9* are expressed more strongly in developing neurons and brain than in adult brain. Thus, their specific over-expression in brain primary tumors supports a functional role for these microRNAs or a link between neuronal stem cells and brain tumorigenesis.

Serum microRNAs are promising novel biomarkers.

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.