Random 6 mers primer

RNA was isolated from TRIzol samples using the TRIzol Reagent following the manufacturer’s instructions. Reverse transcription and Real-time PCR was performed using standard methods as described (Gao et al., 2015 (link)). The first-strand complementary DNA (cDNA) was generated by reverse transcription with Random 6 mers primer (Takara Bio USA, Inc. Mountain View, CA). To quantify the mRNA expression using real-time PCR, aliquots of first-stranded cDNA were amplified with gene-specific primers and Power SYBR Green PCR Master Mix (Bio-Rad) using a StepOne Real-Time PCR System (Applied Biosystems). The PCR reactions contained cDNA, Universal Master Mix (Applied Biosystems), and 10 mM of forward and reverse primers in a final reaction volume of 20 ml. The mRNA expression of different samples was calculated using 2^{-deltadelta Ct} method (Livak and Schmittgen, 2001 (link)).