title: A real-time one-step reverse transcriptase-polymerase chain reaction method to quantify c-erbB-2 expression in human breast cancer.
abstract: We developed a real-time one-step reverse transcriptase-polymerase chain reaction (RT-PCR) method for the routine quantification of c-erbB-2 oncogene expression in breast cancer, using a 7700 ABI PRISM Sequence Detector System (Perkin Elmer-Applied Biosystems, Courtaboeuf, France). The real-time quantification of the polymerase chain reaction products is based on the TaqMan 5' nuclease assay. The optimal experimental conditions we determined were as follows: 6 mM MgCl2, 200 nM of fluorogenic probe, 200 nM of each primer, and 12.5 units MuLV reverse transcriptase. The GAPDH housekeeping gene was used for normalization of c-erbB-2 expression. In human breast cancer cell lines, the normalized expression of c-erbB-2 ranged from 8 x 10(-6) to 2,600 x 10(-6), the two highest values corresponding to the c-erbB-2 overexpressing cells MDA-MB-453 and SK-BR-3. In a series of 100 breast cancer samples, c-erbB-2 normalized expression was found to range from 0.4 x 10(-6) to 350 x 10(-6). A close correlation was observed between this real-time one-step quantitative RT-PCR method and both semiquantitative conventional RT-PCR (N = 22; r = 0.8543; P < .0001) and c-erbB-2 protein expression (p185) quantified by an enzyme immunoassay (EIA) (N = 27; r = 0.71; P < .0001). The current realtime RT-PCR assay is rapid, sensitive, and reproducible and appears particularly suitable to quantify gene expression in large series of samples.
authors.affiliation: Laboratoire d'Oncologie Moléculaire Humaine, Centre Oscar Lambret, Lille, France.
authors.forename: V
authors.initials: V
authors.lastname: Pawlowski
authors.forename: F
authors.initials: F
authors.lastname: Révillion
authors.forename: L
authors.initials: L
authors.lastname: Hornez
authors.forename: J P
authors.initials: JP
authors.lastname: Peyrat
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism*; D017931:DNA Primers / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005821:Genetic Techniques; D006801:Humans; D007124:Immunoenzyme Techniques; D015636:Magnesium Chloride / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D017931:DNA Primers; D015636:Magnesium Chloride; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases