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

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


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.

Publication types

  • Comparative Study
  • Evaluation Study
  • Validation Study

MeSH terms

  • Biomarkers, Tumor / genetics
  • Biomarkers, Tumor / metabolism*
  • Cell Line, Tumor
  • Equipment Design
  • Equipment Failure Analysis
  • Gene Expression Profiling / instrumentation*
  • Genes, Reporter / genetics
  • High-Throughput Screening Assays / instrumentation*
  • Humans
  • Miniaturization
  • Neoplasms, Experimental / genetics
  • Neoplasms, Experimental / metabolism*
  • Real-Time Polymerase Chain Reaction / instrumentation*
  • Reproducibility of Results
  • Sensitivity and Specificity


  • Biomarkers, Tumor