Bio-Rad offers robust, one-step reverse transcription (RT)-qPCR reaction formats for increased speed, higher inhibitor tolerance, and optimal PCR efficiency. A proprietary blend of passive reference dyes enables use on any instrument.
iTaq™ universal SYBR® Green one-step kit delivers relative quantification (ΔΔCq) results comparable to two-step RT-qPCR. Terminally differentiated human hepatic cells (HepaRGtm) were induced using one of six known drug metabolism enzyme inducers, omeprazole (250 µM), artesunate (250 µM), rifampicin (250 µM), carbamazepine (250 µM), dexamethasone (15 µM), or β-naphthoflavone (25 µM) in induction media containing a final concentration of 0.1% DMSO. Control samples were incubated with induction media containing 0.1% DMSO. After 72 hours of induction, total RNA was extracted using the Aurum™ total RNA mini kit. One-step RT-qPCR was performed using the iTaq™ universal SYBR® Green one-step kit and 1 ng of total RNA. Two-step RT-qPCR was performed with SsoAdvanced™ SYBR® Green supermix using cDNA prepared with the iScript™ advanced cDNA synthesis kit for RT-qPCR from 1 ng of RNA. PrimePCR™ gene expression assays were used to analyze the fold change in expression of 13 genes, ALB, TF, AFP, CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP3A4, CYP2D6, CYP2E1, GSR, CASP3, and KRT18 relative to three reference genes, RPL13A, TBP, and YWHAZ. Real-time amplification data were collected on a CFX384™ real-time PCR detection system and the gene expression results were analyzed using CFX Manager™ 3.0. Quality control was performed to filter data for all replicate reaction sets with a Cq standard deviation greater than 0.5 or an undetected replicate.
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