Evaluation of housekeeping genes for studies in breast cancer cell lines treated with progesterone

Abstract

Real-time semi-quantitative PCR (qPCR) is extensively used to assess variations in gene expressions resulting from pathological conditions or biological responses. Normalization of qPCR data is required to control innate variations that occur during experimental procedures. Endogenous control genes (ECs), used for normalization of the data should be expressed constitutively and consistently across treatments groups. The aim of the present study was to identify the most suitable endogenous control genes for qPCR based analyses to study responses in breast cancer cell lines following exposure to Progesterone (P4). The expression and validity of five candidate ECs (PUM1, RPS13, RPL13A, TBCA and PSMB) were determined in three breast cancer cell lines following exposure to Progesterone. Gene expression data was analyzed using three methods, Normfinder, Bestkeeper, and Comparative delta Ct method. A significant difference in variance of expression levels of individual ECs under different growth conditions was observed. Expression of PUM1 and PSMB was minimally affected in breast cancer cell lines under the experimental conditions. Indeed, in the progesterone treated cells,PUM1 and PSMB were identified as the most stable EC genes by all three methods while TBCAand RPS13 were least stable. In breast cancer cell lines, our results highlight PSMB as another stably expressed endogenous control gene besides the previously identified PUM1. PSMB gene expression may thus provide an additional parameter for studies in breast cancers.