RealTime ready RT-qPCR Assay Development and Qualification

1 RealTime ready Assay Production and Function Testing 2 Production 2 Function Testing 2 A Census on 10,000+ RealTime ready Assays 6 Selecting the Design Sequence 6 Assay Design 6 Amplification Efficiencies 8 Bibliography References 9 M. Dietrich, U. H. Grepl, B. Kramer and H. Walch Roche Diagnostics GmbH Nonnenwald 2 82377 Penzberg / Germany RealTime ready RT-qPCR Assay Development and Qualification Abstract On the RealTime ready Configurator [1], we offer function tested human, mouse, and rat qPCR assays for gene expression quantification based on the unique Universal ProbeLibrary technology (UPL) [2]. The Universal ProbeLibrary is a set of short hydrolysis probes (8–9-mers) containing modified bases (locked nucleic acids, or LNAs) in order to stabilize the binding and thus ensure high flexibility together with good amplicon specificity [2, 4]. UPL probes are labeled at the 5' end with fluorescein (FAM) and at the 3' end with a dark quencher dye. UPL assays are compatible with all real-time PCR instruments capable of detecting fluorescein (FAM) or SYBR Green I. Each assay is function tested according to stringent criteria [5]. The RealTime ready assays are available as Focus Panels and Custom Panels (pre-plated and dried-down on LightCycler® 480 Multiwell Plates, 96 or 384), or as liquid single assays. Focus Panels contain assays for predefined targets of a specific biochemical pathway, such as apoptosis, cell cycle, etc. Custom Panels are custom configured with RealTime ready assays of choice. Each assay consists of a primer pair (forward and reverse primer) and a UPL probe. The main advantage of RealTime ready RT-qPCR assays is that each individual assay is designed and function tested for specificity, PCR efficiency, and signal intensity according to stringent criteria and in general consistency with the recently published MIQE guidelines [6, 7]. In this document, we describe the processes involved in on-demand RealTime ready RT-qPCR assay development for human, mouse, and rat target genes, and provide an overview on key parameters of more than 10,000 RealTime ready RT-qPCR assays. For life science research only. Not for use in diagnostic procedures. March 2011 Whitepaper RealTime ready Custom RT-qPCR