Primescript rt reagent kit with cdna

The primers used for real-time quantitative reverse transcription (RT)-PCR are depicted in S1 Table. Total RNA was extracted from the liver using the PrimeScript RT reagent Kit with cDNA (TaKaRa, MN, Japan) according to the manufacturer’s instructions. Total RNA (1 μg) was treated with DNase I and used for the RT reaction. The synthesized cDNA was stored at -20°C until further use. Real-time quantitative (q) RT-PCR was performed with the iCycler iQ system (ABI, MN, USA) and the SYBR Green I fluorescence kit (Sigma, MN, USA) according to the manufacturer's instructions. Amplification of mRNAs was performed in duplicate in a 96-well PCR reaction plate (ABI, MN, USA). The following real-time qRT-PCR protocol was used: cDNA was denatured at 95°C for 5 min to activate the Hot-start Taq DNA polymerase. The amplification and quantification program was repeated 40 times (95°C for 30 s, 95°C for 5 s, and 65°C for 31 s).The mRNA expression of GHR, IGF-1, IGFBP-1 and IGFBP-3 was measured using the 2^-△△CT method.