Taqman mir assay protocol

Total RNA was extracted from cell lines 48 h after transient transfection (p. t.) of miR-221 using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). RNA from paraffin-embedded PCa tissues was extracted as described previously [12 (link)]. The RNA concentration was determined with a bioanalyzer (Agilent, Santa Clara, CA, USA). cDNA was synthesized from total RNA with stem-loop reverse transcription primers, according to the TaqMan miR-assay protocol (Life Technologies, Carlsbad, CA, USA) or the Promega ImProm II reverse transcription system (Promega, Madison, WI, USA). Mature miR-expression was quantified with the TaqMan miR-221 assay kit and an Applied Biosystems 7900 HT system. We followed the protocol provided in the manufacturer’s instructions (Life Technologies, Carlsbad, CA, USA). The expression of small nuclear RNA (snRNA) RNU6b was used for normalization. Relative miR expression was calculated with the comparative ∆Ct-method (∆Ct sample = Ct sample − Ct RNU6b). Fold changes in miR expression between samples and controls were determined by the 2^∆∆Ct method (in this study, referred to as the ∆∆Ct method). mRNA expression analysis of VEGFR2/KDR expression was performed according to standard qRT-PCR procedures. Primer sequences are available upon request. Mean Ct was always determined from triplicate PCRs.