2.3. RNA Extraction and Complementary Deoxyribonucleic Acid (cDNA) Synthesis

The total RNAs were isolated from the frozen liver tissues using an RNA isolation kit based on the manufacturer's instructions (Yekta Tajhiz, Iran). The quantity and purity of the extracted RNAs were determined by NanoDrop (Thermo Fisher Scientific, USA) and run in 1% agarose gel. The cDNA synthesis was performed using a reverse transcription kit (Biofact, South Korea) according to the manufacturer's instructions. Briefly, the first strand of cDNA was synthesized in the total volume of 10 μl, containing 1 μl (1 ng) of mRNA, 1 μl of RT enzyme, 4 μl of 10X buffer, 2 μl of random hexamer and poly-A (dT) primers, and 2 μl of the nuclease-free water. Then, the reaction mixture was incubated at 42°C for 1 hour. The cDNA was stored at −80°C for use in real-time polymerase chain reaction (PCR).