Eastep rna extraction kit

RNA was isolated using the Eastep RNA Extraction Kit (Promega, Beijing, China). mRNA was transcribed into cDNA using VigoScript cDNA Synthesis Kit (Vigorous Biotechnology, Shanghai, China). q-PCR was performed using SYBR® Green Master Mix (Thermo Fisher) on a 7300 thermocycler (Invitrogen, Carlsbad, CA, USA). Primer sequences are as follows: TNF-α: 5'-ggagaagggtgaccgactca-3' and 5'-ctgcccagactcggcaa-3'. β-actin: 5'-tcacccacactgtgcccatctacg-3' and 5'-cagcggaaccgctcattgccaatg-3'.

The total RNA of liver was extracted with an Eastep® RNA extraction kit (LS1040, Promega Biotech, China) according to the manufacturer's instruction. The quality of RNA was assessed by a 1.2% agarose gel electrophoresis, and the quantity of total RNA was measured with a spectrophotometer (NP80, IMPLEN, Germany). First- strand cDNA synthesis was performed by using an Eastep® RT Master Mix Kit (LS2050, Promega Biotech, China). β-Actin was used as reference gene after the stability of its expression was confirmed. The specific primers for alkaline phosphatase (akp), acid phosphatase (acp), alanine aminotransferase (alt), and aspartate aminotransferase (ast) were designed by NCBI online tools (Supplementary Table 1). RT-qPCR was performed in a 20 μL reaction volume including 10 μL SYBR Green premix, 0.8 μL cDNA template, 0.4 μL forward and reverse primers (10 μM), and 8.4 μL diethyl pyrocarbonate-treated water. The reaction conditions are as follows: 95°C 30 s, 40 cycles of 95°C 6 s, and 60°C 25 s. After obtaining data, the 2^−ΔΔCt method was employed to calculate gene expression level [25 (link)] and then subjected to statistical analysis.