Messenger RNAs were extracted with TRIzol reagent (Life Technologies Ltd, Carlsbad, CA, USA, cod. 15596018). Complementary DNAs (cDNAs) were prepared with SuperScript™ VILO™ MasterMix (Life Technologies Ltd, Carlsbad, CA, USA, cod. 11755-050) as indicated by the manufacturer. The cDNAs were amplified by PCR in a CFX Connect Real-Time PCR Detection System (BioRad, Hercules, California, USA, cod. 1855201) with the fluorescent double-stranded DNA-binding dye SYBR Green (BioRad, Hercules, California, USA, cod. 1708882). Specific primers for each gene were designed to work under the same cycling conditions (95 °C for 10 min followed by 40 cycles at 95 °C for 15 sec and 60 °C for 1 min), thereby generating products of comparable sizes (about 200–300 bp for each amplification). Primer combinations were positioned whenever possible to span an exon-exon junction and the RNAs were digested with DNAse to avoid genomic DNA interference. Primer sequences are indicated in Supplementary Data 2 . For each reaction, standard curves for reference genes were constructed based on six four-fold serial dilutions of cDNA. All samples were run in triplicate. The template concentration was calculated from the cycle number when the amount of PCR product passed a threshold established in the exponential phase of the PCR. The relative amounts of gene expression were calculated with Cyclophilin-A (CyA) expression as an internal standard (calibrator). The results, expressed as N-fold differences in target gene expression, were determined as follows: N*target = 2(ΔCt sample-ΔCt calibrator).
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