Quantification of Gli1 mRNA

Total RNA was subjected to reverse transcription using qscript cDNA synthesis kit (#95047 Quanta Biosciences) and subjected to qPCR analysis using SYBR-Green (PerfeCTa^® SYBR^® Green FastMix^®, ROX^TM; #95073; Quanta Biosciences) and the following oligonucleotide primers: Gli1 Fwd 5′-CCCATAGGGTCTCGGGTCTCAAAC-3′ and Gli1 Rev 5′-GGAGGACCTGCGGCTGACTGTGTAA-3′ for Gli1 mRNA amplification and Gapdh Fwd 5′-TGGCAAAGTGGAGATTGTTGCC-3′ and Gapdh REV 5′-AAGATGGTGATGGGCTTCCCG-3′ for Gapdh mRNA as an internal control. Equal amounts of RNA were used and reactions were carried out for 40 cycles in StepOne Real-time PCR apparatus (Applied Biosystems). Average relative quantity (RQ) was calculated by the ΔΔCt method.