Identification of endogenous reference genes for qRT-PCR analysis in normal matched breast tumor tissues

Gur-Dedeoglu, B.
Konu, O.
Bozkurt, B.
Ergul, G.
Seckin, S.
Yulug, I. G.
Source Title
Oncology Research
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Electronic ISSN
Cognizant Communication Corporation
353 - 365
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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, TTC22, ZNF224) were determined in 23 primary breast tumors and their matched normal tissues using qRTPCR. 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 qRTPCR 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.

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Book Title
Breast cancer, Endogenous reference genes, Normalization factor, Real-time quantitative RT-PCR, beta 2 microglobulin, beta actin, beta glucuronidase, cyclophilin A, Drosophila protein, gelsolin, glyceraldehyde 3 phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, interleukin 22, interleukin 22 receptor alpha 1, messenger RNA, mitochondrial ribosomal protein L19, phosphoglycerate kinase, polypeptide, porphobilinogen deaminase, Pumilio homolog 1, ribosomal protein L41, ribosomal protein large P0, ribosome protein, ribosome RNA, RNA 18S, succinate dehydrogenase, succinate dehydrogenase complex subunit A, TATA binding protein, tetratricopeptide repeat domain 22, tetratricopeptide repeat protein, tyrosine 3 monooxygenase tryptophan 5 monooxygenase activation protein zeta polypeptide, unclassified drug, zinc finger protein, zinc finger protein 224, tumor marker, adult, aged, article, base pairing, cancer grading, clinical article, controlled study, DNA microarray, down regulation, gene expression, gene targeting, genetic analysis, genetic variability, genomic instability, human, human tissue, molecular cloning, nucleotide sequence, priority journal, reverse transcription polymerase chain reaction, breast tumor, female, gene expression profiling, genetics, reverse transcription polymerase chain reaction, standard, Breast Neoplasms, Female, Gene Expression Profiling, Humans, Reference Standards, Reverse Transcriptase Polymerase Chain Reaction, Tumor Markers, Biological
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