Customized, function tested RealTime ready assays for gene expression analysis in biomarker research and early drug development

Sabine Lohmann1, Andrea Herold1, Bergauer Tobias3, Belousov Anton2, Betzl Gisela1, Dietrich Manuel1, Hessel Harald5, Poignee-Heger Manuela1, Geho David4, Weisser Martin2
1Roche Applied Science, Penzberg, Germany; 2Roche Pharma, Penzberg, Germany; 3Roche Pharma, Basel, Switzerland; 4Roche Pharma, Nutley, USA; 5Ludwig-Maximillian-University, Institue of Pathology, Munich, Germany

Personalized Health care (PHC) uses gene-based information to understand requirements for health maintenance, disease prevention, and therapy, all tailored to an individual’s genetic uniqueness. Biomarkers are therefore a central element throughout a drug’s lifecycle, from target identification to drug application. In biomarker research and early drug development, the investigation of mRNA expression profiles is of great interest. Roche RealTime ready custom panels offer a broad variety of intron-spanning RT-qPCR assays covering the major signal transduction pathways, including those relevant for oncology research. RealTime ready custom panels are comprised of ready-to-use LightCycler® 480 Multiwell Plates containing pre-plated qPCR assays for human, mouse, and rat targets that have been selected by the user. We have established various workflows applicable for gene expression analysis in biomarker research as well as in pre-clinical and early clinical studies. Pre-clinical drug development was started by profiling gene expression using a multi-parameter panel (93 target and 3 housekeeping genes) covering the pathway of interest in a broad screening approach. The goal was to generate a first hypothesis for predictive and pharmacodynamic markers, as well as to provide a workflow with the highest convenience and throughput for this screening approach. This first step was performed in vitro with tumor cell lines treated with the compound of interest. A workflow protocol was developed, starting with an automated RNA extraction on the MagNA Pure LC Instrument in combination with pre-plated RealTime ready customized assays in a 384-well format. The relative gene expression had been analysed. After selecting a set of parameters, this first hypothesis was verified using an in vivo mouse model. Xenograft-derived fresh frozen (FF) tissue samples were used to test for selected biomarkers. Next, we moved to early clinical development using the first set of clinical samples, formalin-fixed paraffin-embedded (FFPE) tissue derived from various tumor entities. Tumor-derived FFPE tissue is the most relevant sample material for the development of therapeutic compounds in clinical trials or in biomarker research. RNA extraction from FFPE tissue (e.g., 10 µm sections) using the High Pure RNA Paraffin Kit, followed by qRT-PCR analysis, is a well established technique; here, we combined this workflow with function tested RTR assays.
For life science research only. Not for use in diagnostic procedures.
LIGHTCYCLER, MAGNA PURE, HIGH PURE, and REALTIME ready are trademarks of Roche.

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