mRNA Expression Profiling
Sabine Lohmann and colleagues from Roche Pharma and Ludwig-Maximillian University discussed the use of customized, function-tested RealTime ready assays for gene-expression analysis in biomarker research and early drug development. They noted that the investigation of mRNA expression profiles in these two areas is of particular interest.
According to the scientists, RealTime ready custom panels offer a variety of intron-spanning RT-qPCR assays covering the major signal transduction pathways, including those relevant for oncology research. The custom panels are composed of ready-to-use LightCycler® 480 multiwell plates containing preplated qPCR assays for human, mouse, and rat targets that have been selected by the user.
The research team explained that they have established various workflows applicable for gene-expression analysis in biomarker research as well as in preclinical and early clinical studies. To illustrate, they said that preclinical drug development was started by profiling gene expression using a multiparameter 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, they added.
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 already had been analyzed. After selecting a set of parameters, this first hypothesis was verified using an in vivo mouse model. Xenograft-derived fresh frozen tissue samples were used to test for selected biomarkers.
The team next moved to early clinical development using the first set of clinical samples, formalin-fixed paraffin-embedded (FFPE) tissue derived from various tumor entities. They explained that 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 was followed by qRT-PCR analysis. Then the researchers combined this workflow with function-tested RTR assays.