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Identification of Novel Endogenous Controls for qPCR Normalization in SK-BR-3 Breast Cancer Cell Line

Laboratory of Molecular Genetics, Institute of Oncology, Faculty of Medicine, Riga Stradiņš University, Dzirciema Street 16, LV-1007 Riga, Latvia
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Academic Editor: Robert Winqvist
Genes 2021, 12(10), 1631; https://doi.org/10.3390/genes12101631
Received: 10 September 2021 / Revised: 12 October 2021 / Accepted: 13 October 2021 / Published: 17 October 2021
(This article belongs to the Section Human Genomics and Genetic Diseases)
Normalization of gene expression using internal controls or reference genes (RGs) has been the method of choice for standardizing the technical variations in reverse transcription quantitative polymerase chain reactions (RT-qPCR). Conventionally, ACTB and GAPDH have been used as reference genes despite evidence from literature discouraging their use. Hence, in the present study we identified and investigated novel reference genes in SK-BR-3, an HER2-enriched breast cancer cell line. Transcriptomic data of 82 HER2-E breast cancer samples from TCGA database were analyzed to identify twelve novel genes with stable expression. Additionally, thirteen RGs from the literature were analyzed. The expression variations of the candidate genes were studied over five successive passages (p) in two parallel cultures S1 and S2 and in acute and chronic hypoxia using various algorithms. Finally, the most stable RGs were selected and validated for normalization of the expression of three genes of interest (GOIs) in normoxia and hypoxia. Our results indicate that HSP90AB1, DAD1, PFN1 and PUM1 can be used in any combination of three (triplets) for optimizing intra- and inter-assay gene expression differences in the SK-BR-3 cell line. Additionally, we discourage the use of conventional RGs (ACTB, GAPDH, RPL13A, RNA18S and RNA28S) as internal controls for RT-qPCR in SK-BR-3 cell line. View Full-Text
Keywords: SK-BR-3; RT-qPCR; reference genes; hypoxia; gene expression; breast cancer cell line; HER2 enriched SK-BR-3; RT-qPCR; reference genes; hypoxia; gene expression; breast cancer cell line; HER2 enriched
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MDPI and ACS Style

Jain, N.; Mitre, I.; Nitisa, D.; Pirsko, V.; Cakstina-Dzerve, I. Identification of Novel Endogenous Controls for qPCR Normalization in SK-BR-3 Breast Cancer Cell Line. Genes 2021, 12, 1631. https://doi.org/10.3390/genes12101631

AMA Style

Jain N, Mitre I, Nitisa D, Pirsko V, Cakstina-Dzerve I. Identification of Novel Endogenous Controls for qPCR Normalization in SK-BR-3 Breast Cancer Cell Line. Genes. 2021; 12(10):1631. https://doi.org/10.3390/genes12101631

Chicago/Turabian Style

Jain, Nityanand, Ingrida Mitre, Dina Nitisa, Valdis Pirsko, and Inese Cakstina-Dzerve. 2021. "Identification of Novel Endogenous Controls for qPCR Normalization in SK-BR-3 Breast Cancer Cell Line" Genes 12, no. 10: 1631. https://doi.org/10.3390/genes12101631

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