Quantitative RNA Expression Analysis

Quantitative RT-PCR RNA extraction and quantitative reverse transcription-PCR (qRT-PCR) were performed as previously described^{3 (link)}. Gapdh was used as the housekeeping gene expression control. Total RNA was isolated from 5 × 10⁶ cells using TRIzol® reagent (Invitrogen, Paisley, UK), according to the manufacturer’s instructions. Complementary DNA (cDNA) was transcribed using SuperScript II Reverse Transcriptase (Invitrogen, Paisley, UK), starting from 1 μg/μl of high pure RNA and samples were tested in triplicate using the SsoFast EvaGreen reagents (Bio-Rad). qRT-PCR protocol was: pre-heating step for 3 minutes at 95 °C, then 40 cycles at 95 °C for 10 seconds and 60° for 30 seconds and last end-step at 65 °C for 10 seconds. Finally, results were analyzed with 2^−ΔΔCt method.