Abstract
BackgroundQuantitative real-time RT-PCR (RT-qPCR) has become a valuable molecular technique in basic and translational biomedical research, and is emerging as an equally valuable clinical tool. Correlation of inter-sample values requires data normalization, which can be accomplished by various means, the most common of which is normalization to internal, stably expressed, reference genes. Recently, such traditionally utilized reference genes as GAPDH and B2M have been found to be regulated in various circumstances in different tissues, emphasizing the need to identify genes independent of factors influencing the tissue, and that are stably expressed within the experimental milieu. In this study, we identified genes for normalization of RT-qPCR data for invasive breast cancer (IBC), with special emphasis on estrogen receptor positive (ER+) IBC, but also examined their applicability to ER- IBC, normal breast tissue and breast cancer cell lines.MethodsThe reference genes investigated by qRT-PCR were RPLP0, TBP, PUM1, ACTB, GUS-B, ABL1, GAPDH and B2M. Biopsies of 18 surgically-excised tissue specimens (11 ER+ IBCs, 4 ER- IBCs, 3 normal breast tissues) and 3 ER+ cell lines were examined and the data analyzed by descriptive statistics, geNorm and NormFinder. In addition, the expression of selected reference genes in laser capture microdissected ER+ IBC cells were compared with that of whole-tissue.ResultsA group of 3 genes, TBP, RPLP0 and PUM1, were identified for both the combined group of human tissue samples (ER+ and ER- IBC and normal breast tissue) and for the invasive cancer samples (ER+ and ER- IBC) by GeNorm, where NormFinder consistently identified PUM1 at the single best gene for all sample combinations.ConclusionThe reference genes of choice when performing RT-qPCR on normal and malignant breast specimens should be either the collected group of 3 genes (TBP, RPLP0 and PUM1) employed as an average, or PUM1 as a single gene.
Highlights
Quantitative real-time RT-PCR (RT-qPCR) has become a valuable molecular technique in basic and translational biomedical research, and is emerging as an valuable clinical tool
We systematically evaluated a panel of endogenously expressed genes to identify those that would be most useful in normalization of Quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) data from Estrogen receptor (ER)+ invasive breast cancer (IBC)
We identified a 3-gene group (TATA-box binding protein (TBP), Ribosomal protein, large, P0 (RPLP0) and homolog of Pumilio, Drosophila, 1 (PUM1)) to be the most suited for normalization of RT-qPCR data in both the collected group of human breast tissue samples (ER+ and ER- IBC and normal breast tissue) and the IBCs (ER+/ ER-)
Summary
Quantitative real-time RT-PCR (RT-qPCR) has become a valuable molecular technique in basic and translational biomedical research, and is emerging as an valuable clinical tool. Prognostic and predictive molecular markers associated with breast cancer are allowing individualization of treatment, and quantitative real-time RT-PCR is frequently used to measure the expression of these markers The advantages of this technique are numerous, including its ability to sensitively quantify specific mRNA despite small samples sizes or low numbers of mRNA [1,2,3,4]. A target gene can be analyzed much more and precisely by correlation to a stable independent parameter, i.e. directly proportional to the amount of mRNA and not influenced by factors such as hormones, cell cycle status, etc This technique is termed 'normalization', and the prevailing method is the use of reference genes [5]. GAPDH continues to be utilized as a normalizer in investigations of breast cancer and cell lines by RT-qPCR
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