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

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.

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

“…To this end, finding cell-specific control genes is recommended before conducting RT-qPCR experiments. [ 12 ] detected that RPL13A was the most stable gene in the consecutive passages of SK-BR-3. As a result, it's critical to verify reference genes before utilizing them in a study, because employing a non-validated reference gene might lead to erroneous conclusions based on faulty data [ 37 ].…”

“…These issues might be solved by employing a stable reference gene to normalize gene expression measurements [ 11 ]. The expression of common reference genes (formerly known as housekeeping genes) like ACTB and GAPDH varies, nevertheless, depending on the type of cell [ 12 ]. These reference genes are essential for the maintenance of basic cellular activities and are expressed constitutively [ 13 ].…”