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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Lyng
Maria B
MB
Department of Pathology, Odense University Hospital, Odense, Denmark. mapetersen@health.sdu.dk
Laenkholm
Anne-Vibeke
AV
Pallisgaard
Niels
N
Ditzel
Henrik J
HJ
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics; D001707:Biopsy, Needle; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D002285:Carcinoma, Intraductal, Noninfiltrating / Q000235:genetics* / Q000473:pathology; D017931:DNA Primers / Q000235:genetics; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D005819:Genetic Markers; D006801:Humans; D007150:Immunohistochemistry; D008875:Middle Aged; D011237:Predictive Value of Tests; D011379:Prognosis; D016601:RNA-Binding Proteins / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D035381:TATA-Binding Protein Associated Factors / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured
D014408:Biomarkers, Tumor; D017931:DNA Primers; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D005819:Genetic Markers; C471242:PUM1 protein, human; D016601:RNA-Binding Proteins; D035381:TATA-Binding Protein Associated Factors