Validation of miniaturized one-step reverse transcription qPCR assays for high-throughput screening and comparison to a reporter gene methodology

Assay and Drug Development Technologies
Catherine BardelleKirsty Rich

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 futur...Continue Reading

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Citations

Jan 23, 2016·Journal of Laboratory Automation·Joe OlechnoLynn Rasmussen
Apr 7, 2017·Tumour Biology : the Journal of the International Society for Oncodevelopmental Biology and Medicine·Ilona HromadnikovaFilip Hron

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Methods Mentioned

BETA
FCS
Assay
PCR

Software Mentioned

GeneData
LightCycler
Roche
GeneData screener
MicroStar

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