Kaempferol-Induced Transcriptional Changes

Kaempferol (1,000 ng/ml; Sigma) was used to stimulate C2C12 cells for 24 h before harvest. TRIzol (Invitrogen Life Technologies, Carlsbad, CA, United States) was used to extract total RNA from cultured cells as previously described (Tang et al., 2012 (link)). RNA purity and integrity were determined by the RNA 6000 Nano assay with an Agilent Bioanalyzer 2,100 (Agilent Technologies, Santa Clara, CA). 1 μg total RNA was reverse transcribed into cDNA using the GoScript Reverse Transcription System (Promega Corp., Madison, WI, United States) based on the manufacturer protocol. qPCR was performed in triplicate using SYBR-Green SuperReal PreMix Plus [Tiangen Biotech (Beijing) Co., Ltd., China] and the iQ5 PCR system (Bio-Rad Laboratories, Inc., Hercules, CA, United States). The reaction conditions were as follows: 95°C for 30 s, and 40 cycles of 95°C for 10 s and 60°C for 30 s. Data were reported as cycle threshold (Ct) values. The 2^−ΔΔCt method was used to compare the RNA expressions. RNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels.