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    Comparison of RNA isolation and associated methods for extracellular RNA detection by high-throughput quantitative polymerase chain reaction

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    Authors
    Tanriverdi, Kahraman
    Kucukural, Alper
    Mikhalev, Ekaterina
    Tanriverdi, Selim E.
    Lee, Rosalind
    Ambros, Victor R.
    Freedman, Jane
    UMass Chan Affiliations
    UMass Metabolic Network
    Program in Molecular Medicine
    Department of Medicine, Division of Cardiovascular Medicine
    Document Type
    Journal Article
    Publication Date
    2016-05-15
    Keywords
    Extracellular RNA
    High-throughput qPCR
    RNA isolation
    RT–qPCR
    miRNA
    Biochemistry
    
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    Link to Full Text
    http://dx.doi.org/10.1016/j.ab.2016.02.019
    Abstract
    MicroRNAs (miRNAs) are small noncoding RNA molecules that function in RNA silencing and posttranscriptional regulation of gene expression. miRNAs in biofluids are being used for clinical diagnosis as well as disease prediction. Efficient and reproducible isolation methods are crucial for extracellular RNA detection. To determine the best methodologies for miRNA detection from plasma, the performance of four RNA extraction kits, including an in-house kit, were determined with miScript miRNA assay technology; all were measured using a high-throughput quantitative polymerase chain reaction (qPCR) platform (BioMark System) with 90 human miRNA assays. In addition, the performances of complementary DNA (cDNA) and preamplification kits for TaqMan miRNA assays and miScript miRNA assays were compared using the same 90 miRNAs on the BioMark System. There were significant quantification cycle (Cq) value differences for the detection of miRNA targets between isolation kits. cDNA, preamplification, and qPCR performances were also varied. In summary, this study demonstrates differences among RNA isolation methods as measured by reverse transcription (RT)-qPCR. Importantly, differences were also noted in cDNA and preamplification performance using TaqMan and miScript. The in-house kit performed better than the other three kits. These findings demonstrate significant variability between isolation and detection methods for low-abundant miRNA detection from biofluids.
    Source
    Anal Biochem. 2016 May 15;501:66-74. doi: 10.1016/j.ab.2016.02.019. Epub 2016 Mar 10. Link to article on publisher's site
    DOI
    10.1016/j.ab.2016.02.019
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/44452
    PubMed ID
    26969789
    Related Resources
    Link to Article in PubMed
    ae974a485f413a2113503eed53cd6c53
    10.1016/j.ab.2016.02.019
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