Thermoscript rt pcr system

Total RNA was extracted from rat liver tissues and HSC-T6 cells using TRIzol reagents (Invitrogen). The first-strand cDNA was synthesized from total RNA using Thermoscript RT-PCR System (Takara) according to the manufacturer's instructions. RT-PCR was carried out under standard protocol using the following primers: β-actin (forward: 5'-TGAGCTGCGTGTGGCCCCTGAG-3'; reverse: 5'-GGGGCATCGGAA CCGCTCATTG-3'), TRPV4(forward: 5'-CGCCTCCGCAGGGATCGCTGGTC-3'; reverse: 5'-TGAGCTGGCTTAGGTGACTCCATGGGAGTG-3'), α-SMA (forward: 5'-TGGCCACTGCTGCTTCCTCTTCTT-3'; reverse: 5'-GGGGCCAGCTTCGTCAT ACTCCT-3'), Col1a1: (forward: 5'-TACAGCACGCTTGTGGATG-3'; reverse: 5'-TT GAGTTTGGGTTGTTGGTC-3'). PCR was performed at 94°C for 5 min, followed by 35 or 38 cycles of amplification at 94°C for 36 s, 52 or 60°C for 36 s and 72°C for 1 min by using ABI9700. The band intensities were measured by a densitometer and the results were normalized with β-actin. The results were repeated at least three times independently from three different pools of templates, while each pool of template was extracted from at least three ventricles.

Total RNA was extracted from human HepG2 cell using TRIzol reagents (Invitrogen). The first strand cDNA was synthesized from total RNA using Thermoscript RT-PCR System (Takara) according to the manufacturer's instructions. RT-PCR was carried out understanding protocol using the primers (Table 1). PCR was performed at 94°C for 5 min, following by 30 or 35 cycles of amplification at 94°C for 40 s, 51 or 60°C for 40 s and 72°C for 1 min by using ABI9700. The band intensities were measured by a densitometer and the results were normalized with β-actin. All experiments were performed in triplicate and repeated at least three times.

RNA was prepared using a NucleoSpin RNA isolation kit (Macherey–Nagel), and cDNA was synthesized using the ThermoScript RT-PCR system (Takara). Quantitative real time RT-PCR was performed using a Thermal Cycler Dice Real Time System with SYBR Premix Ex Taq II (Takara). Relative mRNA expression levels of target genes were calculated using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Primers are listed in Supplementary Table 2.