Prism 7700 sequence detector

Total RNA was extracted from subcutaneous adipose tissue using TRIzol (Takara Biotechnology, Dalian, China). Total RNA with A260:A280 ratio above 1.85 was used for real-time PCR analyses. First strand cDNA was reverse-transcribed from 1 μg of total RNA by using a SuperScript first-strand synthesis kit (Takara) according to the manufacturer’s instruction. Quantification of murine ADP and β-actin mRNA was performed with a PRISM 7700 sequence detector (Life Technologies, Shanghai, China), by using TaqMan probe. The primers were designed by Takara Biotechnology. PCR cycling conditions were as follows: 2 min at 50 °C, 10 min at 95 °C followed by 40 cycles of denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 1 min. Amplification was performed in a final volume of 25 µL, and each sample contained 3 µL cDNA (equivalent to 100 ng of RNA), 900 nmol of specific primers, 225 nmol of the probe, and a master mix made from quantitative PVR core kit (Takara). The mean value of Ct for each sample was used for data analysis. All samples were normalized to the β-actin values and the results expressed as fold changes of Ct changes of Ct value relative to controls by using the 2^{−ΔΔ Ct} formula.