Faststart universal sybr green master pcr kit

RNA isolation from the liver tissue samples (~20 mg) was carried out using the RNeasy Plus mini kit (Qiagen, Valencia, CA) as per the manufacturer’s instruction and was reverse-transcribed (1 μg total RNA) using a High Capacity RNA-to-cDNA^™ Kit (Life Technologies, Grand Island, NY). Real-time PCR was performed in a final volume of 10 μl containing 50 ng of cDNA template and specific sets of forward and reverse primers using a FastStart Universal SYBR Green Master PCR Kit (Roche Applied Science, Indianapolis, IN) and an ABI Prism 7700 system (Applied Biosystems^® Life Technologies, Grand Island, NY) according to the manufacturer’s protocols. The oligonucleotide primers and their sequences used in RT-PCR are shown in Table 1. The amplification process time was as follows: 95°C for 2 min; 40 cycles at 95°C for 20 s, 60°C for 20 s, 72°C for 30 s, and a final 5-min extension at 72°C. Results were normalized to the housekeeping gene 36B4, and the values of the control (chow-fed) group were set to 1.