Quantification of relative expression of genes with homologous sequences using fluorescence-based single-strand conformation polymorphism analysis--application to lactate dehydrogenase and cyclooxygenase isozymes.
The combination of reverse transcribed-PCR and fluorescence-based single strand conformation polymorphism analysis has been proposed for the quantitative determination of ratio of mRNA molecules with homologous sequences. We applied this procedure to lactate dehydrogenase subunits M and H, and cyclooxygenase 1 and 2. We designed fluorescence labeled common PCR primers in the sequences highly homologous between two subunit and isozyme cDNAs, and performed reverse transcribed-PCR and fluorescence-based single strand conformation polymorphism analysis. PCR efficiency was almost the same for the different target sequences, so analysis of mixtures of known amounts of lactate dehydrogenase M and H revealed linear and precise proportions of lactate dehydrogenase M mRNA. It was shown that template concentrations and number of PCR cycles did not affect the determination of proportions of lactate dehydrogenase M to total lactate dehydrogenase. The procedure was applicable to a determination of cyclooxygenase-2 proportion; furthermore, the present procedure could be easily applied to investigation of expression levels of genes encoding mRNAs with homologous sequences.
Maekawa
M
M
Division of Clinical Laboratory, National Cancer Center Hospital, Tokyo, Japan. mmaekawa@gan2.ncc.go.jp
Sugano
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D003402:Creatine Kinase / Q000378:metabolism; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; D018076:DNA, Complementary; D005453:Fluorescence; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000235:genetics*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D018807:Polymorphism, Single-Stranded Conformational; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D007527:Isoenzymes; D008565:Membrane Proteins; D012333:RNA, Messenger; D007770:L-Lactate Dehydrogenase; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C496511:PTGS1 protein, human; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D003402:Creatine Kinase