Revertra ace rt pcr kit

Total RNA was prepared by RNA-TRIZOL extraction (Gibco). Concentration and purity of the extracted RNA were measured spectrophotometrically at A260 and A280. Real-time reverse transcription-polymerase chain reaction (real-time PCR) was performed using TOYOBO ReverTra Ace RT-PCR kit according to the manufacturer's instructions. Primers were F:5′-GTGGGAGCGACAACTTTACC-3′/ R:5′-GAGAACGAAACCAGGGCTACT-3′ for Sox9; F:5′-aCAAGAGCAAGGGAA GAAGCA-3′/R:5′-TGGACAGTAGACGGAGGAAAG-3′ for Collagen type II; F:5′-A GAATCCATAACTGCCCCAAC-3′/R:5′-GTCACGCCC TCCACTAACTCT-3′ for Aggrecan; F:5′-GAAGGTGAAGGTCGGAGTC-3′/R:5′-GAAGATGGTGATGGGA TTTC-3′ for GAPDH; F:5′-ATGGAGTTGCGTGTTGGA-3′/R:5′-GTCAACATACA ACACTTTCTG-3′ for CK1ε and F:5′-CGCTTTGCTGAGGTCTATAAGGC-3′/R:5′-GATATTGGAGCTCTTGAGGTCCCT-3′ for TGFRII. A typical reaction (50 μL) contained 1/50 of reverse transcription-generated cDNA and 200 nM of primer in 1 × SYBR Green RealTime Master Mix (Toyobo, Shanghai, China) buffer. The PCR reactions were carried out on a Bio-Rad IQ5 multicolour detection system using 2 μg of synthesized cDNA under the following conditions: 95°C for 5 minutes, 40 cycles at 95°C for 15 seconds, 60°C for 15 seconds and 72°C for 30 seconds. All real-time PCRs were performed at least in triplicate. The value was always normalized to the control group.