塔克曼
RNA提取
小RNA
核糖核酸
互补DNA
实时聚合酶链反应
分子生物学
生物
聚合酶链反应
逆转录酶
逆转录聚合酶链式反应
基因表达
计算生物学
基因
遗传学
作者
Kahraman Tanrıverdi,Alper Küçükural,Ekaterina Mikhalev,Selim E. Tanriverdi,Rosalind Lee,Victor Ambros,Jane E. Freedman
标识
DOI:10.1016/j.ab.2016.02.019
摘要
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.
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