2.8 Reverse transcription q-PCR validation of DEGs
In this work, to elucidate the validity of the RNA-seq data,
quantitative reverse-transcription polymerase chain reaction (q-RT-PCR)
was performed for some of the DEGs. The information of primers is
presented in Table 4. Total RNA remaining from RNA-seq library
preparation was utilized for q-RT-PCR. RNA was reverse transcribed into
cDNA utilizing M-MLV Reverse Transcriptase (Vazyme). Real-time PCR was
performed with the StepOne RealTime PCR System using the SYBR Green PCR
Reagents Kit (Yeasen). The PCR conditions compose of denaturing at 95˚C
for 10 min, 40 cycles of denaturing at 95˚C for 15 s, annealing and
extension at 60˚C for 1 min. For each sample, PCR amplifications were
performed in triplicate. The RNA expression levels of all the genes were
standardized against that of GAPDH.