title: Validation of miniaturized one-step reverse transcription qPCR assays for high-throughput screening and comparison to a reporter gene methodology.
abstract: Quantitative real-time polymerase chain reaction (PCR) is regarded as the gold standard for molecular profiling and target identification, but not in the context of high-throughput screening owing to limitations on workflow, cost of reagents, and miniaturization opportunities. Recent advances have moved reverse transcription quantitative PCR (RT-qPCR) forward, such as improvements in liquid handling, the launch of higher throughput platforms, and the release of one-step products. These one-step reagents enable the user to go straight from a cellular assay format to qPCR without the need for cumbersome and potentially expensive multistep RNA purification protocols. Our aim was to investigate the use of a one-step accelerated workflow to measure the levels of epidermal growth factor receptor (EGFR) and nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) gene expression using lysates generated by the RealTime ready Cell Lysis kit in downstream quantitative RT-qPCR. We present, for the first time, data from a vendor-independent one-step 1536 workflow that compares reporter gene and RT-qPCR screening approaches for oncology drug discovery. We also demonstrate a miniaturized and high-throughput workflow that could enable future application of this sensitive assay technology, with particular impact against phenotypic assays and those using rare cell types.
authors.affiliation: Discovery Sciences iMed, AstraZeneca, Global HTS Centre , Macclesfield, Cheshire, United Kingdom .
authors.forename: Catherine
authors.initials: C
authors.lastname: Bardelle
authors.forename: Lisa
authors.initials: L
authors.lastname: McWilliams
authors.forename: Susan
authors.initials: S
authors.lastname: Mounfield
authors.forename: Mark
authors.initials: M
authors.lastname: Wigglesworth
authors.forename: Kirsty
authors.initials: K
authors.lastname: Rich
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004867:Equipment Design; D019544:Equipment Failure Analysis; D020869:Gene Expression Profiling / Q000295:instrumentation*; D017930:Genes, Reporter / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000295:instrumentation*; D006801:Humans; D008904:Miniaturization; D009374:Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D014408:Biomarkers, Tumor