Validating Gene Expression Data by QRT-PCR

In order to verify the reproducibility and repeatability of gene expression data obtained by RNA-Seq, seven genes were selected for QRT PCR verification. In brief, cDNA was generated from total RNA using the PrimeScript II 1 st Strand cDNA Synthesis Kit (Takara, Dalian, China) following the manufacturer’s instruction. Quantitative RT-PCR analysis was carried out with the cDNA using SYBR green on a Roche LightCycler 96 real-time PCR machine (Roche, Basel, Switzerland). The b-Actin was used as a reference gene for the standardization of the results. The relative expression levels were calculated as described previously (15 (link)). Three biological repeats were measured for each sample. The primers used were shown in Table 2.