Effect of hepatocyte growth factor and angiotensin II on rat cardiomyocyte hypertrophy

Chen, Ai-Lan; Ou, Cai-Wen; He, Zhao-Chu; Liu, Qi-Cai; Dong, Qi; Chen, Min-Sheng

After the cells were treated with HGF (10 ng/mL), Ang II (1 µM), or HGF (10 ng/mL) plus Ang II (1 µM) for 12 h, they were washed with ice-cold PBS buffer. Total RNA was extracted from the cells using Trizol (21) , and 1 µg RNA was reverse transcribed to cDNA using Thermo ScriptRT-PCR (22) . Real-time PCR was performed using an Applied Biosystems 7500 fast real-time PCR system and SYBR Premix Ex Taq mixture with specific primers (Sangon, China) (23) . The PCR assays were initiated with a denaturation step at 95°C for 10 s, followed by amplification with 40 cycles at 95°C for 10 s and annealing at 60°C for 20 s www.bjournal.com.br Braz J Med Biol Res 45 (12) 2012 (two-step method) (24) . Finally, a melting curve analysis was performed from 60° to 85°C. Data were evaluated with the Applied Biosystems software. The following primers designed with the Primer Express Software (Applied Biosystems) were used: ANF: 5'-AAA GCA AAC TGA GGG CT-3' (sense) and 5'-GGG ATC TTT TGC GAT CT-3' (antisense); β-MHC: 5'-TGC AGT TAA AGG TGA AGG C-3' (sense) and 5'-CAG GGC TTC ACA GGC AT-3' (antisense); glyceraldehyde 3-phosphate dehydrogenase (GAPDH): 5'-CGG AGT CAA CGG ATT TGG TGG TAT-3' (sense) and 5'-AGC CTT CTC CAT GGT GGT GAA GAC-3' (antisense). GAPDH was used as an internal control and the amount of target was defined by the 2 ∆∆Ct method (25) .