Amv reverse transcriptase reverse transcription system

For all the RT and q-PCR experiments, total RNA was isolated from each sample using miRNeasy Mini Kit (Qiagen, Valencia, CA) according to manufacturer's manual and quantitated using NanoDrop1000 spectrometry (Thermo Scientific, Wilmington, DE). Equal amounts (1μg) of total RNA were reverse transcribed using Oligo dt primer and AMV reverse transcriptase (Reverse Transcription System, Promega, Madison, WI). 10 ng of total RNA per 15 μl reaction was reverse transcribed using TaqMan miR reverse transcription kit with specific miR primers (Applied Biosystems, Foster City, CA). Q-PCR was performed in triplicate with specific (Survivin, CUG-BP1, HuR, miR-214-3p, U6 and GAPDH) TaqMan primers and probes (Applied Biosystems, Foster City, CA). Two μl cDNA were used in total 20 μl volume per reaction. Reactions were run on a STEP-ONE Plus Real–Time PCR System (Applied Biosystems, Foster City, CA) and cycling conditions were as follows: 2 min at 50°C, 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. The threshold limit was set so that it intersected all the samples during the log-liner phase of amplification. The levels of GAPDH were used to normalize levels of survivin, CUG-BP1, and HuR in q-PCR samples. For miR experiments, normalization was accomplished using small nuclear RNA U6.