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

Nityanand Jain,Ingrida Mitre,Valdis Pirsko,Dina Nitisa,Inese Cakstina-Dzerve

doi:10.3390/genes12101631

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, 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.

Highlights

Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) represents a modified variant of the popular conventional PCR with diverse applications, ranging from functional genomics to molecular medicine, virology, microbiology, and biotechnology [1]
Our study reports for the first time to our knowledge, a comprehensive analysis, combining previous and novel candidates studied over multiple successive passages (p), in replicate cultures S1 and S2, and validated in various hypoxic conditions for SK-BR-3 cell line
Novel candidate reference genes were selected on the basis of HER-E breast cancer sample transcriptomic data from TCGA legacy dataset by applying the following criteria: (I) medium to high expression levels—mean (log2(TPM) ≥ 5; (II) low expression variance—standard deviation (log2(TPM)) ≤ 1; (III) no exceptional expression—no log2 (TPM) differs from the mean log2 (TPM) by a factor of two or more [71]

Summary

Introduction

Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) represents a modified variant of the popular conventional PCR with diverse applications, ranging from functional genomics to molecular medicine, virology, microbiology, and biotechnology [1]. Large-scale analysis of expression patterns is performed by RNA-Seq or high-throughput microarray analysis, the findings for individual genes usually are validated by RT-qPCR due to its high sensitivity, specificity, reproducibility, and broad dynamic range [2,3,4]. This enhanced sensitivity of RT-qPCR imposes special conditions. Other considerations include standardization of RT-qPCR protocols [5], maintaining consistency of used reagents [6,7]

Methods

Results

Conclusion