Gene Selection Using PrimePCR Plates And CFX Maestro Software

Quantitative real-time PCR is one of the most important methods for rapid and reliable quantification of gene expression. In order to perform an accurate analysis of relative gene expression in different samples, tissues, or experimental conditions, reference genes must be used to normalize variation across all samples. While the importance of choosing the appropriate reference genes has been widely recognized, researchers often rely on knowledge over experience and often simply choose to use classical reference genes (GAPDH, B2M, ACTB, TBP, and the like). Although these commonly used reference genes may be stable in many situations, it has been observed that their expression can vary considerably depending on species, genotype, developmental stage, experimental treatment, or circadian rhythm (Jain et al. 2018, Lacerda et al. 2014, Radonic et al. 2004). Additionally, reference genes reported in the literature for a particular sample type should be validated experimentally prior to use. Some sources of variability that must be included in the reference gene stability survey include sample type and treatment(s). Other experimental sources of variability can be minimized by making sure to harvest cells, prepare and quantitate RNA, and synthesize cDNA using the same methods and reagents. It is important to evaluate several reference genes and select only those that are stably expressed across all samples and sets of conditions. Use of multiple stably expressed reference genes for normalization can further reduce gene expression (Vandesompele et al. 2002).