Sybr green real time pcr master

Total RNA including miRNA fraction was collected from ASMCs using a TRIzol-based extraction protocol (Invitrogen). Isolated RNAs were polyadenylated using the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Logan, UT, United States). cDNA synthesized was used to perform quantitative PCR on an IQ™5 Multicolor Real-Time PCR Detection System (Bio-Rad) using the SYBR Green Real-time PCR Master (TaKaRa, Tokyo, Japan). Primers specific for miR-206 and U6 small nuclear RNA were purchased from Sangon Biotech (Shanghai, China), the following primer sets were used: rat miR-206, RT primer, 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCACAC-3′, Forward, 5′-GCGGCGGTTCACAGTGGCTAAG-3′, Reverse, 5′-ATCCAGTGCAGGGTCCGAGG-3′; U6, RT primer, 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATA-3′, Forward, 5′-AGAGAAGATTAGCATGGCCCCTG -3′, Reverse, 5′-ATCCAGTGCAGGGTCCGAGG-3′. The fold increase relative to control samples was determined by 2^−ΔΔCt method. The miRNA expression was normalized to U6 small nuclear RNA. Amplification was performed at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 58 °C for 30 s and 72 °C for 30 s.

Total RNA was extracted from PASMCs using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. cDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Logan, UT, USA). The cDNA synthesized was used to perform quantitative PCR on an IQ™5 Multicolor Real-Time PCR Detection System (Bio-Rad, Richmond, CA, USA) using the SYBR Green Real-time PCR Master (TaKaRa, Tokyo, Japan). Primers for TRPC6 and β-actin were purchased from Sangon Biotech (Shanghai, China). Primers were as follows: TRPC6, Forward, 5′-GGTGCGGAAGATGCTAGAAG-3′, Reverse, 5′-AATTTCCAGGTGCTCATTGG-3′; β-actin, Forward, 5′-ACGGTCAGGTCATCACTATCGGCAATG-3′, Reverse, 5′-ACAGCACTGTGTTGGCATAGAGGTCTT-3′. Amplification was performed at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 58 °C for 30 s and 72 °C for 30 s. The fold increase relative to control samples was determined by 2^−ΔΔCt method. The expression of TRPC6 was normalized to β-actin.