4.12. Quantitative RT-PCR

Total cellular RNA was isolated from mouse hepatocytes, 3T3-L1 cells, adipose tissue and liver using TRIzol reagent as per the manufacturer's instructions. Reverse transcription was performed with a high-capacity complementary DNA reverse transcription kit (Applied Biosystems, Foster City, CA). Quantitative reverse transcriptase-PCR was then performed on a 7300 Real-Time PCR system (Applied Biosystems) using SYBR Green Master Mix (Applied Biosystems) as per the manufacturer's instructions. Beta actin was used as the endogenous control. The PCR amplifications were examined using the 7300 Real-Time PCR system operated SDS version 1.4 program and Delta Rn analysis method (Applied Biosystems). The primers used for quantitative reverse transcriptase-PCR are shown in Table 1.