Ali Rihani1, Tom Van Maerken1, Barbara D’haene2, Nurten Yigit1, Jo Vandesompele1,2, Rita Rein3
1Center for Medical Genetics, Ghent University, Ghent, Belgium; 2Biogazelle, Ghent, Belgium; 3Roche Diagnostics, Germany
Roche’s RealTime ready Focus Panels are ready-to-use, pre-validated qPCR assays targeting selected genes from specific pathways or functional groups. We aimed to evaluate the utility of the technology for convenient and reliable gene expression analysis in accordance to the MIQE guidelines. A pilot experiment was set up using the human apoptosis panel that enables expression profiling of 372 genes. Two previously established chemosensitive and chemoresistant neuroblastoma cell lines were analyzed before and after treatment with the MDM2 inhibitor nutlin-3. Thorough post-qPCR quality control and data-processing was performed using Biogazelle’s qbasePLUS software. We were able to demonstrate (1) the expression stability of a selected subset of the included reference genes, (2) the utility of the integrated positive and negative RT controls and RNA integrity assessment, (4) the ease and power of data analysis using qbasePLUS, and (3) the power of the strategy to obtain biologically relevant results when scrutinizing an entire pathway. Overall, the study demonstrates that the RealTime ready Focus Panels in combination with the state-of-the-art real-time PCR data-analysis software qbasePLUS enable easy, fast, and reliable gene expression quantification.
|Back to MIQE and QC strategies in qPCR|