Quantifying miR-15a and miR-16 Expression

For quantification of endogenous or transfected miR-15a and miR-16 expression levels total RNA was isolated using TRI Pure reagent (Roche, Indianapolis, IN 46250-0414 USA), quantified, 1 μg RNA was reverse transcribed and quantitative polymerase chain reaction was performed using TaqMan® miRNA assays (Cat No. 000389 for hsa-miR-15a, Cat No. 000391 for hsa-miR-16, and Cat. No 001973 for U6; from Life Technologies, Grand Island, NY, US). Relative miR expression levels were calculated using the comparative C_t method with U6 as the normalizer [6 (link)].

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Dhar Dwivedi S.K., Mustafi S.B., Mangala L.S., Jiang D., Pradeep S., Rodriguez-Aguayo C., Ling H., Ivan C., Mukherjee P., Calin G.A., Lopez-Berestein G., Sood A.K, & Bhattacharya R. (2016). Therapeutic evaluation of microRNA-15a and microRNA-16 in ovarian cancer. Oncotarget, 7(12), 15093-15104.

Publication 2016

TRI Pure reagent

Assays Mirna Polymerase chain reaction

Corresponding Organization : University of Oklahoma

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Variable analysis

independent variables

Expression levels of miR-15a and miR-16

dependent variables

Relative expression levels of miR-15a and miR-16 normalized to U6

control variables

Total RNA isolation using TRI Pure reagent
Reverse transcription of 1 μg RNA
Quantitative polymerase chain reaction using TaqMan® miRNA assays

controls

U6 as the normalizer for relative miR expression levels

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