ArticlePLoS computational biology2022
RNA-Seq is not required to determine stable reference genes for qPCR normalization.
Article in PLoS computational biology, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed, 13 citations in OpenAlex.
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- Enhancing gene expression studies in bovine embryos fertilized in vitro: Identifying stable reference genes across blastocysts with different developmental speeds.The Journal of reproduction and development · 2025Article
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- Comparative Root Transcriptome Profiling and Gene Regulatory Network Analysis between Eastern and Western Carrot (Plants (Basel, Switzerland) · 2023Article
- C-Type Natriuretic Peptide Acts as a Microorganism-Activated Regulator of the Skin CommensalsBiology · 2023Article
- Transcriptome-based selection and validation of optimal reference genes in perirenal adipose developing of goat (Frontiers in veterinary science · 2022Article
- Article
Corrections and comments
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Authors and funding
12 authors at 3 institutions in 4 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Assessment of differential gene expression by qPCR is heavily influenced by the choice of reference genes. Although numerous statistical approaches have been proposed to determine the best reference genes, they can give rise to conflicting results depending on experimental conditions. 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. We validated these observations in two distinct cross-sectional experimental conditions involving human iPSC derived microglial cells and mouse sciatic nerves. 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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.