Iscript reverse transcription kit

RNA was extracted using TRIzol RNA Isolation method (Invitrogen) following the provider's instructions. cDNA was synthesized using iScript reverse transcription kit (Biorad). For real time qPCR performed with SYBRgreen (Biorad), primers for the following genes were used: fgf21, 5-GCTCTCTATGGATCGCCTCA-3 (forward) and 5-TTGTAACCGTCCTCCAGCAG-3 (reverse); fas, 5- CACAGATGATGACAGGAGATGG-3 (forward) and 5- TCGGAGTGAGGCTGGGTTGAT-3 (reverse); β-actin, 5-GATGTATGAAGGCTTTGGTC-3 (forward) and 5-TGTGCACTTTTATTGGTCTC-3 (reverse). For real time qPCR performed with TaqMan (Thermo Scientific), the following probes were used: fgf21, Mm00840165_g1; β-actin, Mm00607939_s1. Real-time qPCR was performed using 25–50 ng of cDNA on a Roche Lightcycler. Target gene expression level was calculated using the ΔΔC_T method, with β-actin as a reference gene, and normalized according to the control condition.

Total RNA from cell cultures was extracted using QIAamp RNA extraction kit (Qiagen, Valencia, CA, United States). cDNA was generated from 500 ng of total RNA with iScript Reverse Transcription Kit (Bio-Rad Laboratories, Hercules, CA, United States). Quantification of gene expression was conducted using iQ SYBR Green Supermix (Bio-Rad Laboratories). The reverse transcription (RT) product was used to measure the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a positive housekeeping gene, AFP, TIMP-1, TIMP-2, TIMP-3, P53, RB, hTERT and c-Myc using real time PCR (qPCR) and specific primer sequences (Table 1). The reaction conditions were as follows: pre-denaturation at 95 ˚C for 3 min; 40 cycles of denaturation at 94 ˚C for 20 s, annealing and elongation at 60 ˚C for 60 s. The threshold cycle (Ct) value for triplicate reactions was averaged, and the relative genomic expression was calculated by the 2^-∆∆Ct method [∆Ct= Ct (gene) - Ct (GAPDH)][25 (link)]. Melting curves were performed to ensure that only a single product was amplified.