Quantitative analysis of Her-2/neu (ERBB2) gene expression using reverse transcriptase polymerase chain reaction.
Inappropriate expression of Her-2/neu (ERBB2) gene has been associated with impaired breast cancer prognosis, suggesting a functional role in tumor progression. Herein we describe a quantitative method for analysis of Her-2/neu gene messenger RNA (mRNA), which employs reverse transcriptase polymerase chain reaction (RT-PCR) on a 10-microns cryostat section. The technique combines modified RNA extraction with complementary DNA (cDNA) synthesis to achieve a high level of sensitivity. Utilizing this PCR-based gene expression assay, we were able to quantitate variable amounts of Her-2/neu mRNA in cell lines with established levels of gene expression and in clinical human breast cancer specimens. In clinical samples, mRNA levels correlated with intensity of immunoperoxidase staining for corresponding oncoprotein. We conclude that PCR-based mRNA quantitation can be applied to quantitative analysis of Her-2/neu gene expression, and potentially many other genes, in samples of limited size.
Sarkar
F H
FH
Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan.
Visscher
D W
DW
Crissman
J D
JD
D001483:Base Sequence; D015139:Blotting, Southern; D001943:Breast Neoplasms / Q000235:genetics*; D004273:DNA, Neoplasm; D006801:Humans; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D018719:Erb-b2 Receptor Tyrosine Kinases; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D018719:Erb-b2 Receptor Tyrosine Kinases