Rotor gene q qrt pcr instrument

qPCR was used for gene expression studies to determine pluripotency (Oct3/4, Sox2, and Nanog), apoptosis (Bax, Bak, p53, Bcl2, and Birc), differentiation (FABP4, PPARγ, ON, OCN, COL2, and COL10A1), and the expression of hypoxia-related genes (GLUT1, LDHA, LOX, and PGK1). Three replicates of each sample were analyzed by qPCR. Relevant primer information is displayed in Table S2. The total RNA was extracted using an RNeasy Minikit (Qiagen, CA, USA) and quantified using an OPTIZEN 3220 UV BIO spectrophotometer (Mecasys, Sungnam, Korea). Next, cDNA synthesis was performed from 1 μg total RNA using an Omniscript Reverse Transcription Kit (Qiagen) with a oligo dT primers at 60°C for 1 h. qRT-PCR was performed using a Rotor Gene Q qRT-PCR instrument (Qiagen) with Rotor-Gene 2× SYBR Green mix (Qiagen), 2 μL cDNA per reaction, and 0.5 mM forward and reverse primers. The qPCR program settings included of pre-denaturation (95°C for 10 min), 45 PCR cycles (95°C for 10 s, 60°C for 6s, and 72°C for 4 s), melting curve analysis (60°C to 95°C ramp, 1°C per seceond ramp rate) and cooling (40°C for 30 s). Transcript levels of all target genes were normalized against those of TBP, which is a stable reference gene in human MSCs [12 (link)].