Ecodry cdna synthesis premix

Total RNA was extracted from liver tissues using RNAiso Plus (Takara Bio Inc., Shiga, Japan) and the first strand of cDNA was synthesized via reverse transcription (EcoDry cDNA Synthesis Premix; Takara Bio, Inc.). The cDNA was amplified with primers and SYBR green (Roche Applied Science, Mannheim, Germany) using a thermocycler (Lightcycler Nano; Roche Applied Science). Gene specific primers used for cDNA amplification were 5′-AGCCCACGTCGTAGCAAACCACCAA-3′ (sense) and 5′-ACACCCATTCCCTTCACAGAGCAAT-3′ (anti-sense) for TNF-α; 5′-GAAAGTCAACTCCATCTGCC-3′ (sense) and 5′-CATAGCACACTACGTTTGCC-3′ (anti ense) for IL-6; and 5′-GGCTGTATTCCCCTCCATCG-3′ (sense) and 5′-CCAGTTGGTAACAATGCCATGT-3′ (antisense) for β-actin. Real-time RT-PCR was performed with an specific amplification cycling conditions as follows: 35 cycles of 30 s at 94°C, 30 s at 65°C and 30 s at 72°C for TNF-α; 35 cycles of 30 s at 94°C, 45 s at 58°C and 30 s at 72°C for IL-6; and 35 cycles of 30 s at 94°C, 30 s at 55°C and 30 s at 72°C for β-actin. The levels of mRNA expression were normalized to that of the β-actin mRNA expression and reported relative to the average of all Δcycle threshold (Ct)-values in each sample using the Ct method. All samples were carried out in duplicate to ensure amplification integrity.