{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,10]],"date-time":"2026-04-10T20:14:22Z","timestamp":1775852062147,"version":"3.50.1"},"update-to":[{"DOI":"10.1371\/journal.pcbi.1009868","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2022,3,10]],"date-time":"2022-03-10T00:00:00Z","timestamp":1646870400000}}],"reference-count":39,"publisher":"Public Library of Science (PLoS)","issue":"2","license":[{"start":{"date-parts":[[2022,2,28]],"date-time":"2022-02-28T00:00:00Z","timestamp":1646006400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["www.ploscompbiol.org"],"crossmark-restriction":false},"short-container-title":["PLoS Comput Biol"],"abstract":"<jats:p>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 \u2018stable\u2019 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.<\/jats:p>","DOI":"10.1371\/journal.pcbi.1009868","type":"journal-article","created":{"date-parts":[[2022,2,28]],"date-time":"2022-02-28T13:34:59Z","timestamp":1646055299000},"page":"e1009868","update-policy":"https:\/\/doi.org\/10.1371\/journal.pcbi.corrections_policy","source":"Crossref","is-referenced-by-count":14,"title":["RNA-Seq is not required to determine stable reference genes for qPCR normalization"],"prefix":"10.1371","volume":"18","author":[{"given":"Nirmal Kumar","family":"Sampathkumar","sequence":"first","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6024-6959","authenticated-orcid":true,"given":"Venkat Krishnan","family":"Sundaram","sequence":"additional","affiliation":[]},{"given":"Prakroothi S.","family":"Danthi","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2371-9109","authenticated-orcid":true,"given":"Rasha","family":"Barakat","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2239-0447","authenticated-orcid":true,"given":"Shiden","family":"Solomon","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2973-4633","authenticated-orcid":true,"given":"Mrityunjoy","family":"Mondal","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3849-8866","authenticated-orcid":true,"given":"Ivo","family":"Carre","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6989-4132","authenticated-orcid":true,"given":"Tatiana","family":"El Jalkh","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7797-9155","authenticated-orcid":true,"given":"A\u00efda","family":"Padilla-Ferrer","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1897-1329","authenticated-orcid":true,"given":"Julien","family":"Grenier","sequence":"additional","affiliation":[]},{"given":"Charbel","family":"Massaad","sequence":"additional","affiliation":[]},{"given":"Jacqueline C.","family":"Mitchell","sequence":"additional","affiliation":[]}],"member":"340","published-online":{"date-parts":[[2022,2,28]]},"reference":[{"key":"pcbi.1009868.ref001","author":"SA Bustin","year":"2009","journal-title":"The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments"},{"key":"pcbi.1009868.ref002","article-title":"Housekeeping genes as internal standards: use and limits","author":"O Thellin","year":"1999","journal-title":"J Biotechnol"},{"key":"pcbi.1009868.ref003","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1016\/j.tig.2013.05.010","article-title":"Human housekeeping genes, revisited","volume":"29","author":"E Eisenberg","year":"2013","journal-title":"Trends Genet"},{"key":"pcbi.1009868.ref004","doi-asserted-by":"crossref","first-page":"e59180","DOI":"10.1371\/journal.pone.0059180","article-title":"Careful Selection of Reference Genes Is Required for Reliable Performance of RT-qPCR in Human Normal and Cancer Cell Lines","volume":"8","author":"F Jacob","year":"2013","journal-title":"PLoS One"},{"key":"pcbi.1009868.ref005","doi-asserted-by":"crossref","first-page":"e0141853","DOI":"10.1371\/journal.pone.0141853","article-title":"With Reference to Reference Genes: A Systematic Review of Endogenous Controls in Gene Expression Studies","volume":"10","author":"JR Chapman","year":"2015","journal-title":"PLoS One"},{"key":"pcbi.1009868.ref006","doi-asserted-by":"crossref","first-page":"RESEARCH0034","DOI":"10.1186\/gb-2002-3-7-research0034","article-title":"Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes","volume":"3","author":"J Vandesompele","year":"2002","journal-title":"Genome Biol"},{"key":"pcbi.1009868.ref007","doi-asserted-by":"crossref","first-page":"509","DOI":"10.1023\/B:BILE.0000019559.84305.47","article-title":"Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations","volume":"26","author":"MW Pfaffl","year":"2004","journal-title":"Biotechnol Lett"},{"key":"pcbi.1009868.ref008","doi-asserted-by":"crossref","first-page":"5245","DOI":"10.1158\/0008-5472.CAN-04-0496","article-title":"Normalization of Real - 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