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

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      Author
      Gur-Dedeoglu, B.
      Konu, O.
      Bozkurt, B.
      Ergul, G.
      Seckin, S.
      Yulug, I. G.
      Date
      2009
      Source Title
      Oncology Research
      Print ISSN
      1555-3906
      Publisher
      Cognizant Communication Corporation
      Volume
      17
      Issue
      8
      Pages
      353 - 365
      Language
      English
      Type
      Article
      Item Usage Stats
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      Abstract
      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.
      Keywords
      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
      Permalink
      http://hdl.handle.net/11693/22506
      Published Version (Please cite this version)
      10.3727/096504009788428460
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      • Department of Molecular Biology and Genetics 442
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