Identification of endogenous reference genes for qRT-PCR analysis in normal matched breast tumor tissues
Author
Gur-Dedeoglu, B.
Konu, O.
Bozkurt, B.
Ergul, G.
Seckin, S.
Yulug, I. G.
Date
2009Source Title
Oncology Research
Print ISSN
1555-3906
Publisher
Cognizant Communication Corporation
Volume
17
Issue
8
Pages
353 - 365
Language
English
Type
ArticleItem Usage Stats
135
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190
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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 cancerEndogenous 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/11693/22506Published Version (Please cite this version)
10.3727/096504009788428460Collections
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