Quantification of miRNA Expression Using TaqMan Assays

The TaqMan miRNA reverse transcriptase kit and TaqMan miRNA assays (Applied Biosystems, Foster City, CA) were used to quantify mature miRNA expression levels. Each target miRNA was quantified according to the manufacturer's protocol with minor modifications. Briefly, reverse transcriptase reactions were performed with miRNA-specific reverse transcriptase primers and 5 ng of purified total RNA for 30 min at 16 °C, 30 min at 42 °C, and finally 5 min at 85 °C to heat-inactivate the reverse transcriptase. All volumes suggested in the manufacturer's protocol were halved, as previously reported [27] (link). Real-time PCRs for each miRNA (10 μl total volumes) were performed in triplicate, and each 10-μl reaction mixture included 2.4 μl of 10×-diluted reverse transcriptase product. Reactions were run on a PRISM 7900HT Fast Real-Time PCR System (Applied Biosystems) at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Twofold dilution series were performed for all target miRNAs to verify the linearity of the assay. To account for possible differences in the amount of starting RNA, all samples were normalized to miR-423. All reactions were run singleplex and quantified using the cycle threshold (ΔΔC_t) method [28] (link).