Identification of genes for normalization of real-time RT-PCR data in breast carcinomas

The 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.A 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.The 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.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 [1-4]. Working with absolute quantities based on standard curves is time-consuming and laborious. A target gene can be analyzed much more easily 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].The primary advantage of using genes expressed within the cells investigated as reference genes is that they also function as endogenous controls, since they are exposed to the same conditions in vivo and in vitro