CFL2基因半定量多重RT-PCR检测方法的建立

Establishment of the Semi-Quantitative Multiple RT-PCR System of CFL2 Gene

目的在CFL2基因功能的研究中,建立一种能检测CFL2基因mRNA表达水平的半定量多重RT-PCR体系。方法细胞总RNA经反转录合成cDNA,以GAPDH为内参与CFL2进行同管扩增,以常规PCR体系为参照对退火温度、Mg^(2+)、dNTP和循环数等参数进行优化并与常规组比较,建立了检测C2C12细胞CFL2基因半定量多重RT—PCR体系。结果CFL2和GAPDH的cDNA同时PCR扩增后电泳条带清晰,与预期产物大小一致,两对引物间无明显竞争,其扩增效率和特异性与单一基因的RT—PCR体系相同。结论成功建立CFL2基因半定量多重RT—PCR方法,有利于半定量检测CFL2 mRNA表达变化。

level in CFL2 gene.Methods cDNA was synthesized with total cell RNA by reverse transcription and amplified together with PCR primers of CFL2 and GAPDH（as internal control）genes.In this system,the annealing temperature,Mg-（2＋） and dNTP concentrations,and cycle number were optimized with reference to the conventional PCR system.Results The result showed that the electrophoresis bands of CFL2 and GAPDH cDNA amplified together by PCR were all clear and their sizes were expected.In our RT-PCR system,there was no obvious competition of amplification between primer sets and the efficiency and specificity of amplification were identical with those in simple gene RT-PCR system.Conclusions A semi-quantitative multiple RT-PCR method was established successfully which would be beneficial to measure the expression level of CFL2 mRNA.