Expression polymerase chain reaction: a sensitive method for analysis of gene expression in human tumours.
To analyse the expression of individual genes in small tumour samples, we have used the method of RNA polymerase chain reaction (PCR) to develop a technique which we have termed expression PCR. With this technique, specific cDNA sequences of a target gene are amplified, analysed by gel electrophoresis, and semi-quantitated using laser densitometry. Alpha-actin is amplified as a reference gene to control for template RNA and each target gene is analysed at several cycle numbers to optimize PCR dynamics. In this study, we have demonstrated expression PCR by analysing the levels of expression of tyrosine kinase genes in a panel of human tumours. We have compared expression PCR with Northern analysis to show that these techniques provide equivalent information on relative levels of gene transcription, with expression PCR requiring 100-fold less RNA. This technique is sufficiently sensitive to detect and compare the levels of expression of genes not seen on Northern analysis and is ideally suited for analysing the expression of multiple genes within the same portion of a tumour.
Cance
W G
WG
Department of Surgery, University of North Carolina at Chapel Hill 27599.
Craven
R J
RJ
Liu
E T
ET
D001483:Base Sequence; D015152:Blotting, Northern; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000235:genetics*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics
D015345:Oligonucleotide Probes; D012334:RNA, Neoplasm; D011505:Protein-Tyrosine Kinases