Rtase cdna synthesis kit

Total RNA was extracted with TRIzol reagent (Invitrogen, USA). miR-23a expression was measured using a TaqMan MicroRNA assay (Ambion Inc., Austin, TX, USA) and was normalized to U6 expression. cDNA was synthesized by a pair of special stem–loop primers, and another pair of primer/probe sets was used to conduct qPCR. The sequences of the miR-23a primers were F: 5’-GCCCATCACATTGCCAGG-3’ and R: 5’-GTGCAGGGTCCGAGGT-3’. To measure CRYAB (alpha B-crystallin) expression, reverse transcription of cDNA from total RNA was conducted with an RTase cDNA Synthesis Kit (Takara, Dalian, China). Quantitative real-time PCR was implemented utilizing SYBR Green PCR Master Mix (Toyobo, Osaka, Japan) with an Applied Biosystem 7900HT system (ABI, Foster City, CA, USA) using the ∆∆C_t method. The CRYAB primer sequences were F: 5’-AACGGCCTGGGTGGATAGAAG-3’ and R: 5’-CAGTACTCACTGAGCTGCTCTT-3’. The primer sequences are shown in Table S1. The CRYAB expression results were normalized to GAPDH expression. All reactions, including those with nontemplate controls, were performed in triplicate.

Cells were lysed and extracted into total RNA using TRIzol (Invitrogen). RNA was reverse transcribed into cDNA using an RTase cDNA Synthesis Kit (Takara, Japan). After that, Applied Biosystem 7900HT System (ABI) was used to quantitatively analyze the mRNA expression of genes by RT-qPCR. Each sample was amplified in a 10 μL reaction volume with specific primers (Supplementary Table S1), SYBR Green PCR Master Mix (Toyobo, Japan) and RNase free water. In this study, GAPDH was applied as an internal control.