Quantifying miRNA Expression by RT-PCR

Real-time analyses by two-step RT–PCR were carried out to quantify miRNA expression using the Thermo Scientific TaqMan chemistry-based miRNA assay system as performed earlier [56 (link)]. 25 ng of cellular RNA were used along with specific primers for human miR-122 (assay ID 000445). U6 snRNA (assay ID 001973) was used as an endogenous Control. One third of the reverse transcription mix was subjected to PCR amplification with TaqMan® Universal PCR Master Mix No AmpErase (Thermo Scientific) and the respective TaqMan® reagents for target miRNA. Samples were analyzed in PCR triplicates from at least two biological replicates. The comparative Ct method which included normalization by the U6 snRNA, was used for each cell type for plotting of mean values with s.d.