Icycler iq optical system software version 3

Quantitative RT-PCR (q-RT-PCR) was carried out in an iCycler™ (Bio-Rad Laboratories) thermocycler using the iScript™ One-Step RT-PCR Kit with SYBR^® Green (Bio-Rad), according to the manufacturer's instructions. Briefly, 5 μl of the extracted sample RNA was added to a 25 μl reaction containing 1X SYBR^® Green RT-PCR Reaction Mix, 300 nM of each primer, and 100 ng of total RNA template. The thermal cycling conditions consisted of a 10 min reverse transcription step at 50°C, 5 min of iScript Reverse transcriptase inactivation at 95°C, followed by 45 cycles of PCR at 95°C denaturation for 10 sec, 60°C of annealing, extension, and detection for 30 sec. Following amplification, melting curves were used to verify the specificity of the PCR product according to its T_m. Melting curve analysis consisted of a denaturation step at 95°C for 1 min, lowered to 55°C for 1 min, and followed by 80 cycles of incubation in which the temperature is increased to 95°C at a rate of 0.5°C/10 sec/cycle. The T_m of each specific PCR product was analyzed using iCycler iQ optical system software version 3.1 (Bio-Rad).