Qinyu Ge

miRNA in plasma exosome is stable under different storage conditions.

Exosomes are small membrane-bound vesicles secreted by most cell types. Exosomes contain various functional proteins, mRNAs and microRNAs (miRNAs) that could be used for diagnostic and therapeutic purposes. How we should store the samples before RNA isolation and whether those long term stored samples could be used for circulating RNA investigation because of RNase is unknown. The aim of the study was to determine the stability of circulating miRNA in exosomes and plasma. Exosomes were isolated from plasma samples by using ExoQuick Precipitation methods. RNA was extracted from exosomes and the corresponding plasma samples with a Qiagen miRNeasy Mini kit. The concentration of RNA was measured by a Qubit® RNA HS Assay Kit, and quantitative PCR was used for individual miRNA expression level detection. Results showed that exosomal miRNA showed extra stability under different storage conditions and no significant influence on plasma miRNA, except for short term storage at 4 °C. It is thus indicated that exosome miRNAs can be good biomarkers based on their stability under various storage conditions.

Next-Generation Sequencing of MicroRNAs for Breast Cancer Detection

It is reported that different microRNA (miRNA) profiles can be detected in the blood of cancer patients. We investigated that whether the key serum miRNAs could discriminate patients with and without breast cancer. This study was divided into three parts: (1) miRNA marker discovery using SOLiD sequencing-based miRNA profiling on cancerous and adjacent noncancerous breast tissue of one breast cancer patient; (2) marker selection and validation by real-time PCR on a small set of serum; (3) gene ontology analysis of the key miRNA target genes. Of genome-wide tissue miRNA expression analysis, five miRNAs were found to be altered more than fivefold by SOLiD sequencing (i.e., miR-29a, miR-23a, miR-23b, miR-192, and miR-21). All the five miRNAs were validated on the 20 breast cancer patients and 20 controls. miR-29a and miR-21 were significantly increased in the serum of breast cancer patients (P < .05). Gene ontology analysis of the target genes revealed enrichment for special biological process categories, that is, signal transduction, development, apoptosis, cell proliferation, and cell adhesion. SOLiD sequencing provides a promising method for cancer-related miRNA profiling. Serum miRNAs may be useful biomarkers for breast cancer detection.

Analysis of serum genome-wide microRNAs for breast cancer detection

OBJECTIVE Among methods for profiling levels of miRNAs, next-generation sequencing (NGS) has an effective one for genome-wide profiles, which not only can accurately quantify known miRNAs expression, but also discovery novel miRNAs. In this paper, we investigated that whether specific miRNAs were co-expressed in the serum and tissue of breast cancer (BC) patients as novel biomarkers by SOLiD sequencing. METHODS Different miRNA expression profiles of serum and tissue in breast cancer patients and control subjects were obtained by NGS -SOLiD sequencing. Real-time PCR was used to selected and validated candidate miRNA-biomarkers. Novel miRNAs were predicted by computational pipeline, and validated by Northern blot analysis. RESULTS Of genome-wide miRNA analysis using SOLiD sequencing, 7 miRNAs were found to be co-upregulated (i.e., miR-103, miR-23a, miR-29a, miR-222, miR-23b, miR-24 and miR-25). miR-222 was significantly increased in the serum of BC patients by further validation(P<0.05), which may be a useful biomarker for differentiating BC patients from controls with receiver operating characteristic (ROC) curve area 0.67 of (95% CI=0.5649 to 0.7775). A novel miRNA, named miR-BS1 was preliminarily identified and validated. Pre-miR-BS1 has a characteristic secondary structure. Mature miR-BS1 expression was detected in MCF-7 and MDA-MB-231 cells. Through gene ontology analysis, predicted target genes of miR-BS1, such as FOXO3 and KRAS, were involved in cancer-related signaling pathway. CONCLUSIONS This study presented a connection between serum- and tissue- based miRNA of breast cancer which suggested that serum-miRNAs may be potential biomarkers for BC detection. And next-generation sequencing will provide a robust platform for miRNA profilings.

A Comprehensive Survey of miRNA Repertoire and 3′ Addition Events in the Placentas of Patients with Pre-Eclampsia from High-Throughput Sequencing

Background To gain insight into potential roles of isomiR spectrum and isomiRs with 3′ additions in pre-eclampsia, we performed a comprehensive survey of miRNA repertoire and 3′ addition events from placental samples with different degrees of pre-eclampsia by applying SOLiD sequencing platform. Principal Findings Over 30% isomiRs were detected with 3′ non-template additional nucleotides, especially for additional nucleotide of adenosine. However, these modified isomiRs showed a lower percentage of total miRNA expression (<15%). Generally, 1-3 abundant isomiRs from a given miRNA locus were identified, but none of them was detected with 3′ additions. Different miRNAs indicated various isomiR spectrums and expression patterns. The most abundant isomiR spectrum, isomiR profile and expression pattern always were stability, but herein we found several exceptions across samples, especially between normal and diseased samples. At isomiR level, we detected a distinct subset of differentially expressed modified isomiRs between normal and diseased samples or between mild and severe samples. Gene Ontology analysis of their experimentally validated target genes revealed enrichment for specific biological process categories. Conclusions The phenomenon of multiple isomiRs, especially for isomiRs with 3′ additions, is not a random event during pre-miRNA processing. Varieties of isomiRs and expression patterns reveal potential functional implication and should be taken into account. The study enriches association of miRNAs and human disease, including potential roles of various miRNA variants and 3′ addition events.

Application of next-generation sequencing technology to profile the circulating microRNAs in the serum of preeclampsia versus normal pregnant women

BACKGROUND Circulating miRNAs, as a new family of miRNAs existing in plasma and serum, had shown great potential to serve as a novel biomarker in body fluid for non-invasive diagnosis and prognosis of plenty kinds of disease, such as cancer and prenatal screening. METHODS In this present study, we analyzed the expression profiles of circulating miRNAs in the serum of four pregnant women with preeclampsia (PE) and one normal control of pregnant women, by the next generation sequencing technology. RESULTS By annotated the raw sequence reads with the databases of miRNA, genome and others small RNA library, miRNA was found to be the major composition of those small RNA-annotated reads. In the result of circulating miRNA profiles in serum, up to 573 distinct miRNAs were annotated to miRBase. The biological features of circulating miRNA in serum were consistent with those tissue/cell based miRNA in the database. Notably, 22 miRNAs were found to be dys-regulated expressed with PE. Compared to the normal control, 15 and 7 miRNAs were up-regulated and down-regulated respectively in each four PE samples. Among these 22 miRNAs, 3 dys-regulated miRNAs have been reported to be dys-regulated in the placentas of PE pregnancies. CONCLUSIONS Results showed that circulating miRNAs in serum of pregnant women could be detected more comprehensive by the next generation sequencing technology. It also suggested that those PE-related miRNAs obtained in this study might be used as notable biomarkers for diagnosis and prognosis of PE.

Maternal Plasma miRNAs Expression in Preeclamptic Pregnancies

Objective. Preeclampsia (PE) is a pregnancy-specific syndrome and one of the leading causes of maternal and fetal morbidity and mortality. The pathophysiological mechanisms of PE remain poorly known. Recently, circulating miRNAs are considered as potential useful noninvasive biomarkers. The aim of this study was to identify differentially expressed plasma miRNAs in preeclamptic pregnancies compared with normal pregnancies. Methods. Maternal plasma miRNA expression profiles were detected by SOLiD sequencing. Differential expressions between mPE/sPE and control group were found. Next, four differentially expressed plasma miRNAs were chosen to validate their expression in other large scale samples by real-time PCR. Results. In terms of sequencing results, we identified that 51 miRNAs were differentially expressed. Four differentially expressed plasma miRNAs (miR-141, miR-144, miR-221, and miR-29a) were selected to validate the sequencing results. RT-PCR data confirmed the reliability of sequencing results. The further statistical analysis showed that maternal plasma miR-141 and miR-29a are significantly overexpressed in mPE (P < 0.05). Maternal plasma miR-144 is significantly underexpressed in mPE and sPE (P < 0.05). Conclusions. Results showed that there were differentially expressed maternal plasma miRNAs in patients with preeclampsia. These plasma miRNAs might be used as notable biomarkers for diagnosis of preeclampsia.

Profiling maternal plasma microRNA expression in early pregnancy to predict gestational diabetes mellitus

To profile the differential expression of plasma miRNAs in gestational diabetes mellitus (GDM).

Deregulated microRNA species in the plasma and placenta of patients with preeclampsia

Emerging evidence indicates that microRNAs (miRNAs), a class of small non-coding RNAs, are involved in a number of biological processes. The results of SOLiD™ sequencing were used to analyze differentially expressed miRNA profiles in the plasma and placenta of patients with preeclampsia (PE) and a subject who had had a pregnancy without complications. miRNAs were identified that were consistently expressed in the placenta, following normalization of the raw data. miRNAs that had increased and differential expression were selected, as defined by percentage >0.02% and a log2 fold change ≥ |1.2|, respectively. This process was repeated in the plasma. Twenty such miRNAs were identified. These were: miR-126, miR-126*, miR-130a, miR-135b, miR-142-3p, miR-149, miR-188-5p, miR-18a, miR-18b, miR-203, miR-205, miR-224, miR-27a, miR-29a, miR-301a, miR-517c, miR-518-3p, miR-518e, miR-519d and miR-93. These miRNAs belonged to 13 clusters or families. However, only four clusters or families involved two or more of these miRNAs. These were the mir-16 cluster, the mir-17 family, the mir-130 family and the mir-517 family. These abnormally-expressed miRNAs and miRNA gene clusters or families are known to be involved in a number of biological processes. Gene enrichment analysis was used to investigate the pathways involved in the development of PE. In conclusion, the miRNAs identified in this study as being abnormally expressed in PE, may be useful as non-invasive diagnostic biomarkers. Co-regulated mRNAs and possible causal pathways involved in the pathogenesis of PE were also identified.

Consistent isomiR expression patterns and 3′ addition events in miRNA gene clusters and families implicate functional and evolutionary relationships

Abstract3′ addition events in miRNAs are widely detected and may contribute to miRNA stability, but little is known about details of the events in miRNA gene clusters and families. Here, we performed a comprehensive analysis of isomiR expression patterns and 3′ additions in miRNA gene clusters and families by analyzing high-throughput sequencing dataset. According to dominant modified isomiRs, miRNA members in many miRNA gene clusters and families showed the same 3′ additional non-template nucleotides. Although clustered miRNAs and homologous miRNAs had consistent or inconsistent expression levels, we found many of them showed consistent expression patterns at isomiR levels. These findings revealed similar processing mechanism and 3′ modification event of miRNAs in gene clusters and families through miRNA maturation process. The consistent maturation mechanism may contribute to co-regulate biological processes, and may originate from ancestral miRNA genes through complex duplication history.

Microarray-in-a-Tube for Detection of Multiple Viruses

Abstract Background: The detection of multiple viruses is important for pathogenic diagnosis and disease control. Microarray detection is a good method, but requires complex procedures for multiple virus detection. Methods: We developed a novel PCR assay, the microarray-in-a-tube system, which integrates multiple PCR processes and DNA microarrays for multiple virus detection. A 5 × 5 oligonucleotide microarray for detecting 4 respiratory tract viruses (severe acute respiratory syndrome–associated coronavirus, influenza A virus, influenza B virus, and enterovirus) with inner controls was arranged on the inner surface of a specially designed Eppendorf cap with a flat, optically transparent window. Results: We were able to perform all detection processes in the encapsulated system without opening the cap. The 4 viruses were successfully amplified by one-step reverse transcription–PCR in the encapsulated tube. After the PCR process, the microarray-in-a-tube was inverted, and the fluorescence-labeled PCR products were directly hybridized on the microarray. Hybridization signals were obtained with an ordinary fluorescent microscope. The sensitivity of the system for virus detection reached 102 copies/μL. With the help of inner controls, the system provided reliable results without false negatives and false positives. Conclusions: The microarray-in-a-tube system is a rapid, labor-saving tool for multiple virus detection with several advantages, such as convenience, prevention of cross-contamination of the PCR products, and potential for multiple-gene detection.