title: Comparison of RNA isolation and associated methods for extracellular RNA detection by high-throughput quantitative polymerase chain reaction.
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.
authors.affiliation: Division of Cardiovascular Medicine, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address: kahraman.tanriverdi@umassmed.edu.
authors.forename: Kahraman
authors.initials: K
authors.lastname: Tanriverdi
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Alper
authors.initials: A
authors.lastname: Kucukural
authors.affiliation: Division of Cardiovascular Medicine, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Mikhalev
authors.affiliation: Division of Cardiovascular Medicine, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Selim E
authors.initials: SE
authors.lastname: Tanriverdi
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Rosalind
authors.initials: R
authors.lastname: Lee
authors.affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Victor R
authors.initials: VR
authors.lastname: Ambros
authors.affiliation: Division of Cardiovascular Medicine, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
authors.forename: Jane E
authors.initials: JE
authors.lastname: Freedman
mesh_terms: D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D018076:DNA, Complementary; D035683:MicroRNAs
keywords: Extracellular RNA; High-throughput qPCR; RNA isolation; RT–qPCR; miRNA