*4.9. Quantitative Reverse Transcription PCR (qRT-PCR) for Measuring mRNA Expression*

Total RNA was reverse transcribed with SuperScript® III First-Strand Synthesis Reverse Transcriptase using a mixture of oligo-d (T) and random hexamer primers (Life Sciences Advance Technology, St. Petersburg, FL, USA). These cDNA preparations were then diluted 2 fold with water before being assayed. qRT-PCR was performed in triplicate in 96-well optical plates with 6 biological replicates. Each reaction contained 1X TaqMan PCR master mix (Applied Biosystems, Waltham, MA, USA with ROX reference dye) and 0.2 µM of each specific primer pair-probe set listed in Table S3. qRT-PCR was performed using a 7500 Fast Real-Time PCR System (Applied Biosystems, Waltham, MA, USA), with

an initial denaturation for 10 minutes at 95 ◦C, primer annealing at 50 ◦C for 2 min, followed by 40 cycles of 15 seconds at 95 ◦C and 1 minute at 60 ◦C. The relative expression of target genes was calculated using the delta-CT method as described [58], and normalized to *GAPDH* expression. The average Ct values were ≤ 30 except for *SOX4* transcripts that showed an average Ct-value of 35 cycles.
