微滴式数字PCR与荧光定量PCR对真皮间充质干细胞增殖及凋亡相关基因检测的比较研究

Comparative Study of Droplet Digital PCR and Real-time Fluorescence Quantitative PCR for the Detection of Proliferation and Apoptosis-related Genes in Dermal Mesenchymal Stem Cell.

目的比较微滴式数字PCR(droplet digital PCR,dd-PCR)和实时荧光定量PCR(real-time fluorescence quantitative PCR,RT-qPCR)对真皮间充质干细胞(dermal mesenchymal stem cell,DMSC)增殖及凋亡相关指标检测的敏感度。方法利用dd-PCR和RT-qPCR分别对寻常型银屑病患者和健康对照者真皮间充质干细胞(dermal mesenchymal stem cell,DMSC)的增殖及凋亡活性相关指标进行检测。结果dd-PCR和RT-qPCR检测结果显示,银屑病DMSC的增殖(PCNA及CDK4)和凋亡活性相关指标(caspase-3)表达水平均较对照组降低,两种方法结果具有高度一致性;但dd-PCR使用的cDNA模板浓度为RT-qPCR的0.01倍。结论对于DMSC分子活性检测而言,dd-PCR和RT-qPCR具有相似的准确性,但dd-PCR敏感度较高,故在模板量受限的情况下,可以优先选择dd-PCR。

Objective To compare the sensitivity between droplet digital PCR(dd-PCR)and real-time fluorescence quantitative PCR(RT-qPCR)on the proliferation and apoptosis-related indexes of dermal mesenchymal stem cell(DMSC).Methods The proliferation and apoptosis-related indexes of dermal mesenchymal stem cell(DMSC)from patients with psoriasis vulgaris and controls were examined using dd-PCR and RT-qPCR,respectively.Results The results of both dd-PCR and RT-qPCR showed the expression levels of proliferation(PCNA and CDK4)and apoptosis-related indicators(caspase-3)of psoriasis DMSC were reduced compared with the control group,and the results of both methods were consistent;however,the concentration of cDNA template used in the dd-PCR method was RT-qPCR 0.01-fold.Conclusion For the detection of molecular activity of DMSC,dd-PCR and RT-qPCR have similar accuracy,but dd-PCR is more sensitive,so dd-PCR can be preferred when the amount of template is limited.