Competitive reverse transcriptase-PCR: an improved method for determination of c-erbB-2 gene expression.
The aim of our study was to establish a simple method for routine analysis of c-erbB-2 gene expression in tumour samples. We constructed a plasmid for the in vitro synthesis of competitor RNA to be used as an internal exogenous control during the RT-PCR detection of c-erbB-2 expression. The competitor RNA harbors a 19-base deletion and 63-base insertion compared to wild-type c-erbB-2 mRNA and generates a PCR product which is easily distinguished from the wild-type PCR product by agarose gel electrophoresis. The sensitivity and reliability of our RT-PCR-system was determined. To address this, we measured c-erbB-2 expression in cultured cells, of which c-erbB-2 expression data were available from Northern blot analysis. In conclusion, our experimental strategy correlated well with the results obtained by Northern blot hybridization, however, it overcomes time consuming and expensive procedures used in classical gene expression analysis.
Schneeberger
C
C
Department of Obstetrics and Gynecology, Molecular Oncology Division, University Hospital of Vienna, Austria.
Eder
S
S
Speiser
P
P
Zeillinger
R
R
D001483:Base Sequence; D001667:Binding, Competitive; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D018734:Genes, erbB-2; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012334:RNA, Neoplasm / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D012334:RNA, Neoplasm; D012194:RNA-Directed DNA Polymerase; D018719:Erb-b2 Receptor Tyrosine Kinases