Gene expression analysis by a competitive and differential PCR with antisense competitors.
We report a sensitive method for the reproducible and accurate measurement of gene expression from small samples of RNA. This method is based on a combination of two PCR techniques: First, an endogenous reporter gene and the gene of interest are simultaneously amplified in one tube after random-primed reverse transcription (RT) of RNA (differential RT-PCR). Second, exogenous homologous fragments of both genes with artificially introduced mutations are added and coamplified in the same reaction (competitive PCR). The first-strand cDNA, and the mutated antisense homologues of the reporter as well as the target gene compete for their respective primers and are therefore amplified with equal efficiencies. After PCR, restriction enzyme digestion allows visualization of the quantitative differences between the four resulting reaction products. The ratios of products that competed during PCR provide the quantitative information. The initial amount of a specific cDNA can be calculated from any competitor/cDNA ratio of reliably measurable PCR product amounts. Extensive competitor titration to experimentally approach the equilibrium is therefore unnecessary. The differential counterpart of competitive and differential RT-PCR (CD-RT-PCR) allows expression of the levels in reference to a reporter gene. MDR1 expression was determined in tumor cells by CD-RT-PCR.
de Kant
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Freie Universität, Berlin, FRG.
Rochlitz
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CF
Herrmann
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D001667:Binding, Competitive; D002294:Carcinoma, Squamous Cell; D016373:DNA, Antisense / Q000032:analysis; D018076:DNA, Complementary / Q000032:analysis; D018432:Drug Resistance, Multiple / Q000235:genetics; D015870:Gene Expression; D008175:Lung Neoplasms; D008433:Mathematics; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D014407:Tumor Cells, Cultured; D001613:beta 2-Microglobulin / Q000235:genetics
D016373:DNA, Antisense; D018076:DNA, Complementary; D012333:RNA, Messenger; D001613:beta 2-Microglobulin