Quantitative Assessment of miRNA Expression

Briefly, total RNA enriched for small RNAs was isolated from whole peripheral venous blood using an mirVana microRNA Isolation Kit (Ambion, Austin, TX, USA). Reverse transcription was performed with a total reaction volume of 10 µL using miRNA-specific stem loop primers and a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Branchburg, NJ, USA) [16 (link),17 (link),18 (link),19 (link)]. Real-time qPCR reactions were performed with a total reaction volume of 15 µL consisting of 3 µL of cDNA, specific primers, TaqMan MGB probes, and the TaqMan Universal PCR Master Mix (Applied Biosystems, Branchburg, NJ, USA) in a 7500 Real-Time PCR System under standard TaqMan PCR conditions [16 (link),17 (link),18 (link),19 (link)].
MicroRNA gene expression was assessed using the delta-delta Ct method as previously described [16 (link),17 (link),18 (link),19 (link),20 (link)]. MicroRNA gene expression data were normalized to the geometric mean of the Ct values of RNU58A and RNU38B, previously selected and tested endogenous controls [21 (link)].