Quantitative Analysis of Gene Expression

RNA was extracted from uninduced and induced i293-ST cells using TRIzol (Invitrogen) (88 (link)). The RNA was DNase treated using the Ambion DNase-free kit according to the manufacturer's instructions, and RNA (1 μg) from each fraction was reverse transcribed with SuperScript II (Invitrogen) according to the manufacturer's instructions, using oligo(dT) primers (Promega). Ten nanograms of cDNA was used as the template in SensiMixPlus SYBR qPCRs (Quantace) according to the manufacturer's instructions, using a Rotor-Gene Q 5plex HRM platform (Qiagen) with a standard 3-step melting program (95°C for 15 s, 60°C for 30 s, and 72°C for 20 s), as previously described (89 (link)). With GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as an internal control mRNA, quantitative analysis was performed using the comparative ΔΔC_T method, as previously described (90 (link)).