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ABSTRACT: The goal of the study was to analyze the microRNAs (miRNA) expression that differentiates in maternal plasma of women before and after parturition. Here, we used high throughput sequencer to analyze the expression change of all microRNAs in plasma from pregnant women. Six families of microRNA were also surveyed using real-time quantitative PCR. Sequencing result showed that the circulating microRNA expression in plasma from pregnant women down regulated remarkably after parturition. The quantitative PCR results showed that the differential expression of most miRNAs in plasma between before and after parturition was consistent with the sequencing result. It is also showed that not only notable differential miRNA expression between the plasma from the same woman collected before and after parturition, but also between normal plasma and preeclamptic plasma, which indicate that miRNAs could be potential biomarker for prenatal diagnosis and prognosis. The study suggested circulating miRNAs in plasma of pregnant women could be detected more comprehensive by the next generation sequencing technology. This research also suggested the differential miRNA expression could be related to the existence and clearance of circulating DNA/RNA, which paved a new way for studying the origin and path of circulating nucleic acid.
Journal of Nanoscience and Nanotechnology 05/2012; 12(5):4035-43. · 1.56 Impact Factor
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ABSTRACT: 3' 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.
Molecular Biology Reports 03/2012; 39(6):6699-706. · 2.93 Impact Factor
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ABSTRACT: The multiplexing becomes the major limitation of the next-generation sequencing (NGS) in application to low complexity samples. Physical space segregation allows limited multiplexing, while the existing barcode approach only permits simultaneously analysis of up to several dozen samples.
Here we introduce pair-barcode sequencing (PBS), an economic and flexible barcoding technique that permits parallel analysis of large-scale multiplexed samples. In two pilot runs using SOLiD sequencer (Applied Biosystems Inc.), 32 independent pair-barcoded miRNA libraries were simultaneously discovered by the combination of 4 unique forward barcodes and 8 unique reverse barcodes. Over 174,000,000 reads were generated and about 64% of them are assigned to both of the barcodes. After mapping all reads to pre-miRNAs in miRBase, different miRNA expression patterns are captured from the two clinical groups. The strong correlation using different barcode pairs and the high consistency of miRNA expression in two independent runs demonstrates that PBS approach is valid.
By employing PBS approach in NGS, large-scale multiplexed pooled samples could be practically analyzed in parallel so that high-throughput sequencing economically meets the requirements of samples which are low sequencing throughput demand.
BMC Genomics 01/2012; 13:43. · 4.07 Impact Factor
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ABSTRACT: Circulating microRNAs in maternal plasma as one type of the cell free nucleotide acid revealed its potential for non-invasive prenatal diagnosis. The next generation sequencing technology provides promising approach detecting miRNA for such purpose.
In this present study, a modified library preparation method for SOLiD sequencing technology was developed and maternal plasma miRNA from single and twin pregnancies was analyzed. Quantitative PCR was carried out for comparison.
Results showed that the sequenced data was improved remarkably with this modified library preparation method; different types and levels of miRNA expression were found in twin pregnancy compared with control. Several miRNAs were validated that remarkably changed in twin pregnancy.
It is indicated that miRNAs might involve the process of pregnancy such as the generation of twin pregnancy, and it also suggested that the specific miRNAs could act as potential biomarkers for clinical diagnosis and therapy.
Clinica chimica acta; international journal of clinical chemistry 10/2011; 412(21-22):1989-94. · 2.54 Impact Factor
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ABSTRACT: 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.
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.
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.
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.
Clinica chimica acta; international journal of clinical chemistry 08/2011; 412(23-24):2167-73. · 2.54 Impact Factor
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ABSTRACT: The small non-coding important regulatory molecules, microRNAs (miRNAs), have been widely and deeply studied especially combining high-throughput sequencing technologies. Here, we attempted to track detailed miRNA precursor metabolic products and gain further insight into pre-miRNA processing by completely analyzing high-throughput sequencing data. Highly expressed miRNA precursors could be entirely covered by various short RNAs and small RNA fragments with a hierarchical distribution. miRNAs and some miRNA* regions were detected quite abundant short RNAs as expected, while other regions of precursors were found shorter RNAs or small fragments with fewer sequence counts. Furthermore, we developed a method to analyze relative expression levels of special RNA classes according to divergence of 5' and 3' ends, respectively. Generally, there were several quite abundant RNA classes from a given miRNA locus, which suggested dominant cleavage sites of Drosha and Dicer during pre-miRNA processing. Compared with 3' end, dominant cleavage site in 5' end always focused on a specific position, which ensured conservation of the identity of miRNA (5'-seed sequence, nucleotides 2-8). Overall, a comprehensive analysis of sequencing data can be used to track pre-miRNA metabolic products and mechanism of pre-miRNA processing and metabolism.
Molecular Biology Reports 06/2011; 39(2):2031-8. · 2.93 Impact Factor
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ABSTRACT: Solid phase minisequencing is an effective single nucleotide determination technique. However, two main methods currently used are both time and money consuming. Here, we introduced a more economical and time efficient approach, called 2-color encoding minisequencing, in which four varieties of nucleotides were labeled by the combination of two fluorescence dyes. Such code was sensitive for different nucleotides, and the sequencing results were highly uniform and repeatable.
Journal of Nanoscience and Nanotechnology 03/2011; 11(3):2305-7. · 1.56 Impact Factor
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ABSTRACT: 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.
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.
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.
PLoS ONE 01/2011; 6(6):e21072. · 4.09 Impact Factor
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ABSTRACT: Toxins produced by bacteria and fungi are one of the most important factors which may cause food contamination. The study of detection methods with high sensitivity and throughput is significant for the protection of food safety. In the present study, we coupled microarray with emulsion PCR and developed a high throughput detection method. Thirteen different gene sites which encode the common toxins of several bacteria and fungi were assayed in parallel in positive and maize samples. Conventional PCR assays were carried out for comparison. The results showed that the developed microarray method had high specificity and sensitivity. Two zearalenone-related genes were investigated in one of the ten maize samples obtained with this present method. The results indicated that the emulsion based microarray detection method was developed successfully and suggested its potential application in multiple gene site detection.
Molecules 01/2011; 16(9):7365-76. · 2.39 Impact Factor
