OGG1基因表达的半定量RT-PCR检测方法的建立

Establishment of a semi-quantitative RT-PCR system for the detection of OGG1 expression

目的培养的犬肾细胞(MDCK)内8-羟基鸟嘌呤DNA糖苷酶(OGG1)基因表达的半定量RT-PCR检测体系的建立。方法培养MDCK细胞后提取总RNA,经优化进行RT-PCR反应,检测细胞内OGG1基因的表达。扩增产物经测序同GenBank中OGG1基因序列比对验证产物的准确性,条带经Image J软件分析并与内参基因甘油醛-3-磷酸脱氢酶(GAPDH)相比,对体系的重复性进行分析。结果扩增产物证实与GenBank中该细胞的OGG1基因序列一致;重复性测定发现,MDCK细胞OGG1与GAPDH灰度值比值的均值为(0.84±0.025),CV值为2.99%。结论本方法的准确性、重复性均较好,可用于半定量检测培养细胞尤其是MDCK细胞内OGG1 mRNA的表达量。

Objective To establish a semi-quantitative RT-PCR system for the detection of the expression of 8-oxoguanine DNA glycosylase （OGG1）in madin-darby canine kidney （MDCK） cells. Methods Total RNA from the cultured MDCK ceils were extracted and used for RT-PCR. The RT-PCR products were sequenced to verify their identities. The expression levels of OGG1 and internal control gene glyceral dehyde-3-phosphate（GAPDH） were analyzed with Image J software to analyze the reproducibility of this method. Results Sequencing results showed that the product is identical with the sequence from the GenBank.The averaged ratio of OGGI/GAPDH was （0.84 ±0.025） and the CV was 2.99%. Conclusion This semi-quantitative RT-PCR method is accurate and reproducible. This method can be used for the detection of OGG1 mRNA from cultured ceils.