Gene Expression Analysis for High Throughput Screening Applications

Albert      Pinhasov; Jay      Mei; Dhammika      Amaratunga; Frank   A.   Amato; Hong      Lu; Jack      Kauffman; Hong      Xin; Douglas   E.   Brenneman; Dana   L.   Johnson; Patricia      Andrade-Gordon; Sergey   E.   Ilyin

Abstract

To meet growing needs for high throughput gene expression profiling, we established a new automated high throughput TaqMan RT-PCR method for quantitative mRNA expression analysis. In this method, the AllegroTM (Zymark) system conducts all sample tracking and liquid handling steps, and ABI PRISM® 7900 HT (Applied Biosystems) is used to conduct real-time determination of the Ct value when amplification of PCR products is first detected and accumulation of inhibitory PCR products is unlikely to occur. The ABI PRISM® 7900 HT Sequence Detection System features a real-time PCR instrument with 384- well-plate compatibility and robotic loading, and continuous wavelength detection, which enables the use of multiple fluorophores in a single reaction. The Allegro System offers an assembly line approach with a modular design that allows reconfiguration of the components to accommodate variations in the assay flow. In the present study, we have established and validated a new automated High Throughput (HT) TaqMan RT-PCRbased method for quantitative mRNA expression analysis. The data demonstrate that HT-Taqman PCR is a powerful tool that can be used for measuring low concentrations of mRNA, and is highly accurate, reproducible, and amenable to high throughput analysis. Results suggest that HT-TaqMan is a reliable method for the quantification of low-expression genes and a powerful tool with HT capability for target identification / validation, structure-activity relationship (SAR) study, compound selection for efficacy studies, and biomarker identification in drug discovery and development.

Keywords: taqman, allegro, rt-pcr, gene expression

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