Firescript cdna synthesis kit

RT-qPCR samples were prepared using commercial kits, according to the manufacturer’s instructions. In brief: total RNA was purified using Quick-DNA/RNA™ Miniprep Kit (Zymo Research, Irvine, CA, USA), 250 ng of purified RNA was reverse transcribed to synthesize cDNA with oligo (dT) primers using FIREScript cDNA Synthesis Kit (Solis BioDyne, Tartu, Estonia), and qPCR was performed with gene specific primers using HOT FIREPol qPCR Mix (Solis BioDyne, Tartu, Estonia) with following conditions: activation 10 min 95 °C; 40 cycles of denaturation 10 s 95 °C; annealing 20 s with temperature optimal for each primer; extension 20 s 72 °C on the RotorGene 6000 system (Corbett Life Science, QIAGEN, Hilden, Germany). Primer sequences are presented in Table S1. mRNA levels were normalized to GAPDH expression. Relative gene expression was calculated using the ΔΔCt method.

RNA was extracted from 264 tumor tissues already preserved in formalin-fixed paraffin-embedded (FFPE) blocks. Sixty independent normal liver tissue specimens were used as controls. Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and quantified using a Nanodrop spectrophotometer (NanoPhotometer Pearl, IMPLEN, Munich, Germany) while considering the samples below 2.0 of the 260/280 ratio. cDNA was synthesized using the Fire Script cDNA Synthesis Kit (Solis Biodyne, Tartu, Estonia) as per the manufacturer’s instructions. PCR was performed with β-actin primers to confirm the cDNA synthesis. Amplified products were electrophoresed on 2% agarose gel and stained with ethidium bromide for further use.