Relative ratios of mRNA molecules encoded by genes with homologous sequences using fluorescence-based single-strand conformation polymorphism analysis.
The combination of reverse transcribed (RT)-PCR and fluorescence-based single-strand conformation polymorphism (SSCP) analysis is proposed for the quantitative determination of the ratio of mRNA molecules with homologous sequences. We applied this procedure to lactate dehydrogenase (LDH) subunits A and B. We designed fluorescence labeled common PCR primers in the sequences highly homologous between LDH-A and LDH-B cDNAs and performed RT-PCR-SSCP analysis. When PCR efficiency was almost the same between the different target sequences, analysis of mixtures of known amounts of LDH-A and LDH-B revealed linear and precise proportions of LDH-A mRNA. Template concentrations and PCR cycles did not affect the determination of proportions of LDH-A to total LDH. 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 Research Institute, Tokyo, Japan.
Sugano
K
K
Ushiama
M
M
Masuda
T
T
Ohkura
H
H
Kakizoe
T
T
Sekiya
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D015179:Colorectal Neoplasms; D017931:DNA Primers; D005091:Exons; D005347:Fibroblasts; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010190:Pancreatic Neoplasms; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D012333:RNA, Messenger / Q000096:biosynthesis / Q000378:metabolism*; D013274:Stomach Neoplasms; D013698:Templates, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D046911:Macromolecular Substances; D012333:RNA, Messenger; D007770:L-Lactate Dehydrogenase