Primescript 1st strand cdna synthesis kit with gdna eraser

Total RNAs were isolated with RNAiso Plus (TaKaRa). For gene expression determination, 500 ng RNA was reverse transcribed into cDNA using PrimeScript 1st Strand cDNA Synthesis Kit with gDNA Eraser (TaKaRa). For miRNA quantification, the cDNA was synthesized using miRNA-specific stem-loop RT primers. The qRT-PCR was performed with a 10 μl reaction volume containing 1 μl of diluted cDNA, 5 μl of UNICON qPCR SYBR Green Master Mix (Low ROX) (Yeasen, ShangHai), 0.4 μl of 10 mM of primers, and 3.2 μl of RNase free water using QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). PCR procedures were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Melting curve analysis was performed at the end to ensure the specificity of each primer pair. The relative expression levels were calculated by the 2^-ΔΔCt method, and α-tubulin and U6 were used as the internal controls of mRNA and miRNA, respectively. The primers used for qRT-PCR are listed in S3 Table.