Quantitative Analysis of Cardiac Gene Expression

The attached cells in each group were selected and digested with pancreatic enzyme (0.25%). Then, 1 ml trizol lysis buffer was added. RNA extraction was carried out by following the manufacture instruction. The A₂₆₀/A₂₈₀ ratio range was from 1.8 to 2.0. The cDNA was synthesized by reverse transcription according to the instruction of PrimeScript RT kits (Takara, Japan).[38 (link)] Amplification and detection of cDNA were performed using a real-time fluorescent quantitative PCR instrument by following the instruction of SYBR qPCR mix (Takara, Japan). Primer sequences of target gene were as follows: SERCA2a Forward primer: 5'-CTAGGCCTCCGGTCCTAACT-3'; and reverse primer: 5'-TGTGAGGAACTGAACCGACG-3'; NCX forward primer: 5'-GGTGAGTGGATTCGGGATCG-3'; and Reverse primer: 5'-CCGTCTCAGCTCTCATGCTT-3'; p47 phox, forward primer: 5'-TCCCAACTACGCAGGTGA AC-3'; and reverse primer: 5'-CCTGGGTTATCTCCTCCC CA-3'; GAPDH forward primer: 5'-ACCACAGTCCATGC CATCAC-3'; and reverse primer: 5'-TCCACCACCCTGTT GCTGTA-3'. Data arrangement and statistical analysis were performed using 2^−ΔΔct analysis method and combined with SPSS16.0 statistical software.