Quantitative PCR for miRNA Expression

Total RNA was extracted from the cell lines and frozen tumor specimens with Trizol reagent (Invitrogen, Calsbad, CA, USA). The RNA was quantified by assessing its absorbance at 260 nm. The cDNA was synthesized from 2 μg of total RNA using M-MLV reverse transcriptase (Invitrogen). As described by Yu [20] (link), stem-loop RT primers were used for the reverse transcription of miRNAs. MicroRNAs were quantitated by real-time PCR using TaqMan MicroRNA assay (Invitrogen, USA). Real-time PCR was performed using a standard TaqMan PCR protocol. The 20 μl PCRs reactions included 1 μl of RT product, 1 Universal TaqMan Master Mix and 1xTaqMan probe/primer mix (Invitrogen, USA, Table S2 in File S1). For miRNAs, U6 snRNA was used as the endogenous control.