Quantification Of Relative Expression Of MRNA With Homologous Sequences Using Single-Strand Conformation Polymorphism Analysis

Purpose: The combination of reverse transcribed(RT)-PCR and single- stand conformation polymorphism (SSCP) analysis is purpose for the quantitative determination of the mRNA molecules with homologous sequences. Methods:â… .Cultivation of the tumor cell and extraction of its total RNA. â…¡.In order to coamplify both LDH-M and LDH-H cDNAs,we designed a set of two PCR primers contained two mismatches to the target sequences of either LDH-M or LDH-H cDNA. â…¢. We utilizated the technic of SSCP to electrophoresis the products of PCR, and analysised the proportion of LDH-M cDNA to the total LDH cDNA and the relation of quantity between LDH-M and LDH-H cDNA. â…£. The lactate dehydrogenase isozyme of tumor cell was detected by a electrophoresis whose support carrier was dacron mambran.Then the isozyme proportion of LDH-M toLDH was calculated out. â…¤.The total protein of tumor cell was detected by coomassie light blue G-250. Results: 1.The PCR cycles and template concentrations did not affect the determination of proportions of LDH-M cDNA to total LDH cDNA. 2.When PCR efficiency was almost the same between the different target sequences, analysis of mixtures of known amounts of LDH-M and LDH-H cDNAs revealed linear and precise proportions of LDH-M cDNA to total LDH cDNA. 3.The proportions of LDH-M to total LDH at mRNA level were almost consistent with those determined by isozyme zymograms at activity level.Conclusion: The present study shows that the combination of reversetranscribed(RT)-PCR and single-strand conformation polymorphism(SSCP) not only could detect the level of mRNA molecules with homologous sequences, but also could overcome the disadvantages of the quantitative analysis to mRNA, such as the disadvantage about PCR efficiency to the end-point assay. The main advantage of our mothod is its simplicity and relatively high sensitivity. The method also lends itself to the simultaneous examination of multipe samples.