Stable reference genes selected from RNAseq data do not offer any significant advantage over conventional reference genes for normalizing qPCR assays

Using robust reference genes which are stably expressed across the samples to normalized qPCR data in a study is crucial. The choice of such reference genes are made based on different approaches. Additionally, a growing body of literature suggests the use of RNA-seq as a prerequisite for choosing optimal reference genes. In this study, we raised two major questions. Firstly, is RNA-seq necessary to find stably expressing genes that could be used as reference genes to normalize qPCR data? In order to address this question we have devised a previously described statistical pipeline of ours to best select the reference genes. Interestingly, we found that RNA-seq is not necessarily needed to find optimal reference genes. Addressing this question raised the second question, is this pipeline is effective for cross-sectional studies and longitudinal studies and in different models of various species. We report that our statistical pipeline that combines two different approaches works robustly in both of the setups we have tried. Comparison of two approaches (stably expressed genes from RNA-seq or conventional genes) to find the optimal reference genes for qPCR analyses.