Dataset Information

A magnetic bead-integrated chip for the large scale manufacture of normalized esiRNAs.

ABSTRACT: The chemically-synthesized siRNA duplex has become a powerful and widely used tool for RNAi loss-of-function studies, but suffers from a high off-target effect problem. Recently, endoribonulease-prepared siRNA (esiRNA) has been shown to be an attractive alternative due to its lower off-target effect and cost effectiveness. However, the current manufacturing method for esiRNA is complicated, mainly in regards to purification and normalization on a large-scale level. In this study, we present a magnetic bead-integrated chip that can immobilize amplification or transcription products on beads and accomplish transcription, digestion, normalization and purification in a robust and convenient manner. This chip is equipped to manufacture ready-to-use esiRNAs on a large-scale level. Silencing specificity and efficiency of these esiRNAs were validated at the transcriptional, translational and functional levels. Manufacture of several normalized esiRNAs in a single well, including those silencing PARP1 and BRCA1, was successfully achieved, and the esiRNAs were subsequently utilized to effectively investigate their synergistic effect on cell viability. A small esiRNA library targeting 68 tyrosine kinase genes was constructed for a loss-of-function study, and four genes were identified in regulating the migration capability of Hela cells. We believe that this approach provides a more robust and cost-effective choice for manufacturing esiRNAs than current approaches, and therefore these heterogeneous RNA strands may have utility in most intensive and extensive applications.

SUBMITTER: Wang Z

PROVIDER: S-EPMC3384639 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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A magnetic bead-integrated chip for the large scale manufacture of normalized esiRNAs.

Wang Zhao Z Huang Huang H Zhang Hanshuo H Sun Changhong C Hao Yang Y Yang Junyu J Fan Yu Y Xi Jianzhong Jeff JJ

PloS one 20120627 6

The chemically-synthesized siRNA duplex has become a powerful and widely used tool for RNAi loss-of-function studies, but suffers from a high off-target effect problem. Recently, endoribonulease-prepared siRNA (esiRNA) has been shown to be an attractive alternative due to its lower off-target effect and cost effectiveness. However, the current manufacturing method for esiRNA is complicated, mainly in regards to purification and normalization on a large-scale level. In this study, we present a ma ...[more]

PMID: 22761791

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Project description:ChIP-seq has become the primary method for identifying in vivo protein-DNA interactions on a genome-wide scale, with nearly 800 publications involving the technique appearing in PubMed as of December 2012. Individually and in aggregate, these data are an important and information-rich resource. However, uncertainties about data quality confound their use by the wider research community. Recently, the Encyclopedia of DNA Elements (ENCODE) project developed and applied metrics to objectively measure ChIP-seq data quality. The ENCODE quality analysis was useful for flagging datasets for closer inspection, eliminating or replacing poor data, and for driving changes in experimental pipelines. There had been no similarly systematic quality analysis of the large and disparate body of published ChIP-seq profiles. Here, we report a uniform analysis of vertebrate transcription factor ChIP-seq datasets in the Gene Expression Omnibus (GEO) repository as of April 1, 2012. The majority (55%) of datasets scored as being highly successful, but a substantial minority (20%) were of apparently poor quality, and another ?25% were of intermediate quality. We discuss how different uses of ChIP-seq data are affected by specific aspects of data quality, and we highlight exceptional instances for which the metric values should not be taken at face value. Unexpectedly, we discovered that a significant subset of control datasets (i.e., no immunoprecipitation and mock immunoprecipitation samples) display an enrichment structure similar to successful ChIP-seq data. This can, in turn, affect peak calling and data interpretation. Published datasets identified here as high-quality comprise a large group that users can draw on for large-scale integrated analysis. In the future, ChIP-seq quality assessment similar to that used here could guide experimentalists at early stages in a study, provide useful input in the publication process, and be used to stratify ChIP-seq data for different community-wide uses.

Project description:Objective: A Tag Array chip was used to detect plasmids of different template concentration, and then analyzed for sensitivity and specificity. Drug resistance genes from tuberculosis clinical specimens were detected, giving comparative phenotypic resistance results to explore the feasibility and value of clinical applications. Methods: Twenty-four strains of Mycobacterium Tuberculosis (MTB) having sequence differences in extracted plasmids of mutant strains. The plasmid was diluted into different concentrations, and then was performed to analyze the sensitivity and specificity of the chip system. A total of 427 clinical specimens (including spinal tuberculosis and pulmonary tuberculosis) were collected from patients who came from seven hospitals. Design, optimization and preparation of the chip detection system, sequencing and phenotypic drug susceptibility results were analyzed to evaluate the sensitivity and specificity of the gene chip. Results: In the template, concentrations of 1 × 103 copies/μL and above were consistent with sequencing results in the mutant. The sensitivity and specificity in spine Tuberculosis specimen of rifampicin (RFP) were 94.40 and 92.86%; isoniazide (INH) were 92.37 and 87.50%; ethambutol (EMB) were 61.36 and 89.29%; fluoroquinolones (FQS) were 79.41 and 92.86%; streptomycin (SM) were 90.18 and 89.29%; second line drugs (SLD) were 77.61 and 83.93%. In Pulmonary Tuberculosis specimen, the sensitivity and specificity respectively were RFP: 92.74%; 93.75%; INH: 91.26%; 87.50%; EMB: 54.17%; 89.58%; FQS: 84.87%; 93.75%; SM: 86.73%; 85.42%; SLD: 80.9%; 91.67%. The RFP, INH, FQs and SM resistance genes was highly sensitive and specific: however, for detection of amikacin (AMK), capreomycin (CPM), kanamycin (KM), specificity was higher, but sensitivity was lower. Sensitivity for the detection of a mutation in the eis promoter region could be improved. Conclusion: Tag Array chip can achieve rapid, accurate detection of tuberculosis resistance, which has important clinical significance and feasibility.

Dataset Information

A magnetic bead-integrated chip for the large scale manufacture of normalized esiRNAs.

Publications

A magnetic bead-integrated chip for the large scale manufacture of normalized esiRNAs.

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