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Assay Drug Dev Technol. 2015 Mar;13(2):94-101. doi: 10.1089/adt.2014.630.

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

Author information

1
Discovery Sciences iMed, AstraZeneca, Global HTS Centre , Macclesfield, Cheshire, United Kingdom .

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.

PMID:
25785772
DOI:
10.1089/adt.2014.630
[Indexed for MEDLINE]

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