Quantitative gene expression measurements from
tumor tissue are frequently compared with matched normal and/or adjacent
tumor tissue expression for diagnostic marker gene selection as well as assessment of the degree of transcriptional deregulation in
cancer. Selection of an appropriate reference gene (RG) or an RG panel, which varies depending on
cancer type, molecular subtypes, and the normal tissues used for interindividual calibration, is crucial for the accurate quantification of gene expression. Several RG panels have been suggested in
breast cancer for making comparisons among
tumor subtypes, cell lines, and benign/malignant
tumors. In this study, expression patterns of 15 widely used endogenous RGs (ACTB,
TBP, GAPDH, SDHA,
HPRT, HMBS, B2M, PPIA, GUSB, YWHAZ2, PGK1, RPLP0, PUM1, MRPL19, and RPL41), and three candidate genes that were selected through analysis of two independent microarray datasets (IL22RA1, TC22, ZNF224) were determined in 23 primary
breast tumors and their matched normal tissues using qRT-PCR. Additionally,
18S rRNA, ACTB, and SDHA were tested using randomly primed cDNAs from 13
breast tumor pairs to assess the rRNA/
mRNA ratio. The
tumors exhibited significantly lower rRNA/
mRNA ratio when compared to their normals, on average. The expression of the studied RGs in
breast tumors did not exhibit differences in terms of grade, ER, or PR status. The stability of RGs was examined based on two different statistical models, namely GeNorm and NormFinder. Among the 18 tested endogenous reference genes, ACTB and SDHA were identified as the most suitable reference genes for the normalization of qRT-PCR data in the analysis of normal matched
tumor breast tissue pairs by both programs. In addition, the expression of the
gelsolin (GSN) gene, a well-known downregulated target in
breast tumors, was analyzed using the two most suitable genes and different RG combinations to validate their effectiveness as a normalization factor (NF). The GSN expression of the
tumors used in this study was significantly lower than that of normals showing the effectivity of using ACTB and SDHA as suitable RGs in this set of
tumor-normal tissue panel. The combinational use of the best performing two RGs (ACTB and SDHA) as a normalization factor can be recommended to minimize sample variability and to increase the accuracy and resolution of gene expression normalization in
tumor-normal paired
breast cancer qRT-PCR studies.