Identification of endogenous reference genes for qRT-PCR analysis in normal matched breast tumor tissues
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.
Endogenous reference genes
Real-time quantitative RT-PCR
beta 2 microglobulin
glyceraldehyde 3 phosphate dehydrogenase
interleukin 22 receptor alpha 1
mitochondrial ribosomal protein L19
Pumilio homolog 1
ribosomal protein L41
ribosomal protein large P0
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
zinc finger protein
zinc finger protein 224
reverse transcription polymerase chain reaction
gene expression profiling
reverse transcription polymerase chain reaction
Gene Expression Profiling
Reverse Transcriptase Polymerase Chain Reaction
Tumor Markers, Biological
Published Version (Please cite this version)10.3727/096504009788428460
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