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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 assay for the expression of 8-oxoguanine DNA glycosylase (OGG1) in canine kidney cells (MDCK). METHODS: MDCK cells were cultured and total RNA was extracted. The expression of OGG1 gene was detected by RT-PCR. The PCR products were sequenced and compared with the OGG1 gene sequences in GenBank to verify the accuracy of the products. The bands were analyzed by Image J software and compared with the internal reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Analyze. Results The amplified product was consistent with the OGG1 gene sequence of GenBank. The repeatability assay showed that the mean gray value of OGG1 and GAPDH in MDCK cells was (0.84 ± 0.025) and the CV value was 2.99%. Conclusion The accuracy and repeatability of this method are good and can be used to semi-quantitatively detect the expression of OGG1 mRNA in cultured cells, especially in MDCK cells.