RNA isolation and qPCR

Total RNA was isolated by using TRIzol or TRIzol LS reagent (Invitrogen) according to the manufacturer’s protocol. miRNA assays by real-time PCR were performed using specific TaqMan primers (Invitrogen). U6 small nuclear RNA (snRNA) was used as an endogenous control. Real-time analyses by two-step RT-PCR were performed for quantification of miRNA levels on a Bio-Rad CFX96 real-time system using an Applied Biosystems TaqMan chemistry-based miRNA assay system. One third of the reverse transcription mix was subjected to PCR amplification with TaqMan universal PCR master mix, no AmpErase (Applied Biosystems) and the respective TaqMan reagents for target miRNAs. Samples were analyzed in triplicates. The comparative Ct method, which included normalization by the U6 snRNA, was used for relative quantification. For quantification of mRNA levels, 200 ng of total cellular RNA was subjected to cDNA preparation followed by qPCR by the SYBR Green method. Each sample was analyzed in triplicates using the comparative Ct method. mRNA levels were normalized with GAPDH as the loading control.