Trizol rna kit

The differentiating cells were treated with 10 ng/ml ghrelin and IGF-1 for 8 days. Total RNA was extracted using a Trizol RNA kit (Promega, United States), and 2 μg RNA was reverse transcribed with an RT-PCR system including M-Mul reverse transcriptase (Promega, United States), RNasin (Promega, United States), Oligo (dT) primer (Promega, United States) and deoxyribonucleoside Triphosphate (TaKaRa, Japan). 2 μl RT products (cDNA) were amplified with human LPL, PPARγ2, C/EBPα, β-actin primers and mouse LPL, IGF-1 primers as shown in Table 1. 10 μl RT-PCR products were electrophoresed in 2% agarose gel in Tris-acetate-EDTA buffer. The gel was then stained with ethidium bromide and photographed using an Alphalmager M2200 (AlphaInnotech, United States). Expression changes were determined by the density ratio of the target genes to mGAPDH or β-actin in 3T3-L1 and human primary preadipocytes, respectively. Experiments were replicated three times with triplicate samples in each experiment.

3T3-L1 preadipocytes were received as a kind gift from the Department of Genetics, Institute of Life Sciences, Peking University, Beijing. Human primary preadipocytes were obtained from the abdominal subcutaneous adipose tissues (SATs) of three patients undergoing surgery (appendicitis, cholelithiasis, and colon benign neoplasm). The clinical details of these three patients are listed in Supplementary Table S1. The study was approved by the ethics committee of Peking Union Medical College Hospital (No. JS-1093). All patients provided informed consent before adipose tissue was obtained during the surgery. Rat ghrelin was purchased from Sigma, United States. Human IGF-1 and IGF-1 antibodies were kind gifts from WHO and NIDDK, NIH, United States, respectively. Trizol RNA kit was purchased from Promega, United States. Ultraviolet-visible spectrophotometer was purchased from Hitachi, Japan. Applied Biosystems PCR System was purchased from Promega, United States.