Isolation of total RNA and quantitative real-time PCR
Total RNA was extracted by TRIzol TM Reagent (Ambion, Texas, USA).
Concentration and purity of RNA were determined using NanoDrop
Microvolume spectrophotometer (Thermo ScientificTM, USA). For miRNA
analysis, 500 ng of total RNA was performed with miRNA 1st Strand cDNA
Synthesis Kits (by stem-loop) (Vazyme, Nanjing, P.R. China) to generate
cDNA. For other mRNA analysis, cDNA was generated by reverse
transcription kit HiScript Q RT SuperMix
(Vazyme, Nanjing, P.R. China).
Quantification of mRNA was done by by real-time PCR performed on
resulting cDNA via miRNA Universal SYBR qPCR Master Mix or ChamQ
Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) and an ABI 7500
thermocycler (Applied Biosystems Life Technologies, Foster City, CA,
USA). The housekeeping gene U6 and GAPDH were used as standards for the
normalization of mRNA expression values. The primers (Sangon Biotech
Co., Ltd., Shanghai, P.R. China) used in this study were listed inTable 2 :