Identification of novel endogenous reference genes in 2D and 3D cultures of MDA-MB-436 breast cancer cell line for RT-QPCR normalization

Objectives. One of the most commonly employed methods to study gene expression is the real time quantitative PCR (RT-qPCR). To avoid sampling errors and adequately interpret the results from qPCR, a panel of reference genes is used, which are thought to have a stable expression across various cell lines, tissues, and conditions. However, recent literature demonstrates that the currently used conventional reference genes might not be as stable as expected. Therefore, identification of more stable reference genes is critical to avoid inaccuracies in normalization and prevent misinterpretation of the results from qPCR. MDA-MB-436 cell line represents triple negative BC and is amongst the most frequently used cell lines in breast cancer research.

Materials and methods. The laboratory protocol was standardized using the MIQE guidelines to limit inter- and intra-replicate variations due to differing methods of preparation. Six biological replicates of MDA-MB-436 cell line were cultured in DMEM/F12, supplemented with 10% FBS, cholera toxin and insulin. Each replicate culture was passaged through six passages and from every second passage six lysates (three lysates – 50% confluence; three lysates – 90% confluence) were collected. We used 34 reference genes for qPCR (13 conventional genes from literature, 12 identified from our previous study in SK-BR-3 cell line, and nine remaining genes were identified using TCGA analysis for TNBC samples). Three genes of interest were used. The collected data were analyzed using NormFinder, geNORM, and RefFinder.

Results. After statistical analysis, the five most stable reference genes were tested, and all normalized the chosen genes of interest as proof of concept.
Conclusions. The heterogeneity in the gene expression across different passages during long-term culturing emphasizes the need for identifying the most stable reference genes to avoid inaccuracies in interpretation of the qPCR results and to allow more precise conclusions.