DM/PK Analysis with iTaq™ Universal SYBR® Green One-Step Kit and PrimePCR™ Assays

Overview of using one-step RT-qPCR with iTaq Universal SYBR Green and PrimePCR assays for drug metabolism enzyme expression analysis. Introduction to the benefits of simplified workflow and screening of Phase 1 enzymes in hepatocyte-like cells.

Grow hepatocyte-like cells in 24-well or 96-well plates and incubate with six test compounds (omeprazole, rifampin, carbamazapine, artesunate, dexamethasone, beta-naphthoflavone) in induction media for 48 to 72 hours.

Extract total RNA using Bio-Rad's Aurum Total RNA Mini Kit for purified RNA, and prepare cell lysates using Bio-Rad's iScript Sample Preparation Reagent for RT-qPCR as an alternative sample preparation method.

Perform reference gene selection using PrimePCR Reference Genes H384 panel on 1 nanogram of purified RNA with iTaq Universal SYBR Green One-Step Kit. Analyze data with qBase Plus to identify three stable reference genes across all treatments.

Perform RT-qPCR on hepatocyte-like cells treated with six compounds using PrimePCR assays for seven cytochrome P450 genes and albumin. Collect data on CFX 384 real-time PCR instrument and calculate relative normalized expression using CFX Manager.

Test three selected compounds (omeprazole, carbamazapine, rifampicin) across a 4,166-fold concentration range on hepatocyte-like cells. Analyze expression patterns of target genes to determine optimal induction concentrations and identify non-specific effects at high doses.

Calculate relative quantification results from the screening experiment using both one-step RT-qPCR and traditional two-step approaches (with RNA purification). Verify data concordance between methods to confirm comparable performance of the simplified one-step workflow.