RNA-Seq is not required to determine stable reference genes for qPCR normalization.Assessment of differential gene expression by qPCR is heavily influenced by the choice of reference genes.  Hence, recent studies propose the use of RNA-Seq to identify stable genes followed by the application of different statistical approaches to determine the best set of reference genes for qPCR data normalization.  In this study, however, we demonstrate that the statistical approach to determine the best reference genes from commonly used conventional candidates is more important than the preselection of 'stable' candidates from RNA-Seq data.  Using a qPCR data normalization workflow that we have previously established; we show that qPCR data normalization using conventional reference genes render the same results as stable reference genes selected from RNA-Seq data.  These results taken together show that given a robust statistical approach for reference gene selection, stable genes selected from RNA-Seq data do not offer any significant advantage over commonly used reference genes for normalizing qPCR assays.
RNA-Seq
RNA-Seq is not required to determine stable reference genes for qPCR normalization.Assessment of differential gene expression by qPCR is heavily influenced by the choice of reference genes.  Hence, recent studies propose the use of RNA-Seq to identify stable genes followed by the application of different statistical approaches to determine the best set of reference genes for qPCR data normalization.  Using a qPCR data normalization workflow that we have previously established; we show that qPCR data normalization using conventional reference genes render the same results as stable reference genes selected from RNA-Seq data.  These results taken together show that given a robust statistical approach for reference gene selection, stable genes selected from RNA-Seq data do not offer any significant advantage over commonly used reference genes for normalizing qPCR assays.
qPCR