Validating Gene Expression by Quantitative PCR

For qPCR validation, the RNA from the three replicates for every condition was pooled. Reverse transcription to cDNA was performed with the qScript cDNA Synthesis Kit (Quantabio, Beverly, MA, USA) A qPCR analysis was performed with the LightCycler 480 SYBR Green I Master mix (Roche, Ref. 04707516001) following the manufacturer’s instructions on the Lightcycler 480 instrument II system (Roche, Basel, Switzerland). The three (YWHAZ, TBP, PLOD1) out of six most stable reference genes were selected with the geNorm method of Vandesompele et al. [51 (link)] and the normalization factor was determined by the geometric mean. All primers for the qPCR reactions can be found in Table S3. The relative expression (compared to the untreated control) of each gene was calculated using the (primer efficiency)^ΔCt method.