Biorad icycler iq sybr green mix

Total RNA was harvested by using the Rneasy Kit (Qiagen, Valencia, CA, USA). CDNA synthesis was done with Superscript II (40 U/l, Invitrogen, Groningen, The Netherlands). Real-time PCR was performed using the Biorad-iCycler IQ detector system and Biorad-iCycler IQ SYBR Green mix (Biorad, Munich, Germany). The PCR primers (Sigma Chemical Company, St. Louis, MO, USA) were: Shh 5'-CAGCGACTTCCTCACTTTCC-3' (forward) and 5'-GGAGCGGTTAGGGCTACTCT-3' (reverse); GAPDH: 5'-ACAGTCAGCCGCATCTTCTT-3' (forward) and 5'-ACGACCAAATCCGTTGACTC-3' (reverse). Fluorescent threshold values were measured in triplicate, and fold changes were calculated by the formula 2-(sample 1 ΔCt - sample 2 ΔCt), where ΔCt is the difference between the amplification fluorescence thresholds of the mRNA of interest and the GAPDH mRNA.