*4.4. Real Time PCR*

The cDNA for miRNA was synthesized from total RNA. Reverse transcription of specific mature miRNAs was performed using TaqMan miRNA assays according to the manufacturer's protocol (ABI). The PCR amplification and reading were conducted in triplicate using the StepOnePlus thermal cycler (ABI). Mature miRNA expression was quantified under the following thermal cycler conditions: 2 min at 50 ◦C, 10 min at 95 ◦C and 40 amplification cycles (15 s at 95 ◦C and 1 min at 60 ◦C). Expression values were calculated based on the comparative threshold cycle (Ct) method. U6 snRNA was used for miRNA levels normalization.
