Quantification of miRNA Expression

Total RNA was extracted from cultured cells, using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. For the preparation of cellular miRNAs, small-sized RNAs containing miRNAs were isolated from total RNA, using the RNeasy MinElute Cleanup kit (Qiagen, Valencia, CA, USA). To evaluate miRNA expression levels, we used single-tube TaqMan miRNA assays as previously described (Applied Biosytems, Franklin Lakes, NJ, USA) (29 (link), 30 (link)). Normalization was performed with 18S. All reverse transcription reactions, no-template controls, and RT primer minus controls were run in a GeneAmp PCR 9700 Thermocycler (Applied Biosystems). Gene expression levels were quantified using the ABI 7300 Real-Time PCR System (Applied Biosytems). Comparative real-time PCR was performed in triplicate, including the no-template controls, using specific primers for miR-9, with all reagents obtained from Applied Biosytems. The TaqMan RNU6B endogenous control kit was purchased from Applied Biosystems. PCR was carried out at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative expression was calculated using the comparative threshold (C_t) method.