Taqman rt qpcr assay

miR-21, 92, 885, 450, 1226, 16 and 1202 expression was validated using the commercially available pre-designed TaqMan RT-qPCR assay (Applied Biosystems, Foster City, CA, USA). The RNA extraction procedure was similar to the above description, and the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) was used to prepare the cDNA. The reverse transcription reactions (20 µL) were performed at 16°C for 30 min, 42°C for 30 min, and at 85°C for 5 min to terminate the reaction. Each reaction included 20 ng of total RNA, multiple (heptaplex) stem-loop miRNA-specific primers from the TaqMan MicroRNA Assays (12.5 nM each), 2 mM dNTPs, 100 U MultiScribe™ Reverse Transcriptase, 1X Reverse Transcription buffer, and 5 U RNase Inhibitor. The hsa-miRs target sequences were amplified in a 10 μL reaction with 5 μL of TaqMan Universal PCR Master Mix, 0.5 μL from the TaqMan miRNA expression assays, 1 μL of cDNA and 3.5 μL of DNase-free water. The PCR cycling conditions were as follows: 2 min at 50°C, 10 min at 95°C, 40 cycles at 15 sec and 95°C and 1 min at 60°C. RNU43 was the endogenous control. The ΔΔCT method was used to calculate the relative miRNA expression: ΔΔCT = (PC sample CT miRNA - PC sample CT endogenous control) - (BPH sample CT miRNA - BPH sample CT endogenous control). The expression fold change was calculated as 2^-ΔΔCT.