Quantitative RT-PCR

mRNA expression levels of adipogenic differentiation related genes at 0, 7, 14 and 21 days were detected by real-time quantitative PCR (qRT-PCR). Total RNA was isolated from the cells using TRIZOL^® Reagent, 500ng total RNA was used for cDNA synthesis using cDNA reverse-transcription kit. Gene-specific primers were designed using Primer Express software. The primer sequences were shown in Table 1. Quantitative real-time PCR was performed by using the Takara kit (SYBR^®Premix Ex TaqTM II, item no. RR820A) according to manufacturer instructions. GAPDH was selected as the internal reference gene, and the PCR reaction was carried out using the ABI 7500HT Fast Real-Time PCR system (Applied Biosystems, Foster City, CA). Relative quantification of mRNA levels was plotted as the fold change using the 2^{− ΔΔ Ct} method, generally compared with those cultured in the control medium. Analyses were performed in duplicates, and all experiments were repeated at least three times.

Primer Sequences Used for Quantitative Real-Time PCR