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

Overview

Journal Genes (Basel)

Publisher MDPI

Date 2021 Oct 23

PMID 34681026

Citations 1

Authors

Nityanand Jain

Ingrida Mitre

Dina Nitisa

Valdis Pirsko

Inese Cakstina-Dzerve

Affiliations

Soon will be listed here.

Abstract

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, and 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 , , and 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 (, , , and ) as internal controls for RT-qPCR in SK-BR-3 cell line.

Citing Articles

Advanced breast cancer diagnosis: Multiplex RT-qPCR for precise typing and angiogenesis profiling.

Awrahman H, Mohamad D Biochem Biophys Rep. 2024; 37:101615.

PMID: 38205186 PMC: 10776909. DOI: 10.1016/j.bbrep.2023.101615.

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