卵泡液中游离线粒体DNA提取和定量方法的研究

Study on extraction and quantification methodfor free mitochondrial DNA in follicular fluid

目的探讨影响卵泡液中游离线粒体DNA(cfmtDNA)提取和定量的因素即卵泡液处理和冷冻储存、实时荧光定量PCR(Q-PCR)引物和试剂盒选择。方法收集2016年3月~10月在华中科技大学同济医学院生殖医学中心进行IVF/ICSI的78名患者的卵泡液样本,分别用4种方法(一步离心法、两步离心法、两步离心法的基础上0.22μm及0.45μm过滤器过滤)对样本进行处理。处理后的样本一部分继续后续操作,一部分冷冻储存后再进行相应实验。用硅胶膜离心柱法,(如TIANamp Genomic DNA试剂盒)和磁珠法(如BeaverBeadsTM Circulating DNA试剂盒)分别提取样本DNA,进行Q-PCR,比较线粒体编码的基因ND1和hmito3为引物cfmtDNA定量的差异及相关性。结果一步离心法获得的cfmtDNA浓度显著高于其余三种方法(P<0.05),其余三种方法之间浓度无显著性差异(P>0.05);与冷冻前相比,冷冻后cfmtDNA浓度无显著性差异(P>0.05);ND1引物扩增cfmtDNA所得的浓度平均值显著高于hmito3引物扩增cfmtDNA所得的浓度平均值(P<0.05),ND1引物和hmito3引物扩增cfmtDNA所得的浓度显著相关(r=0.63,P<0.000 1);与TIANamp Genomic DNA试剂盒相比较,BeaverBeadsTMCirculating DNA试剂盒提取cfmtDNA的量显著增加(P<0.05)。结论卵泡液样本的处理方法、引物和试剂盒的选择都会影响卵泡液中cfmtDNA的定量。建议选用两步离心法处理卵泡液,磁珠法提取游离DNA,ND1引物进行cfmtDNA的定量。

DNA（cfmtDNA）in follicular fluid, namely follicular fluid treatment and cryopreservation, selection of real time fluorescence quantitative PCR（Q -PCR）primers and kit. Methods： Follicular fluid samples from 78 patients with IVF/ICSI in Tongji Medical Reproductive Medical Center of Huazhong University of Science and Technology from March to October 2016 were treated with four different methods： one step centrifugation, two step centrifugation, two step centrifugation plus filtration with 0.22 μm or 0.45 μm filter. Then one part of the processed samples continued to do the subsequent experiments, and another part of samples subjected to the corresponding experiment after froze storage. DNAs of the sample were extracted with TIANamp Genomic DNA Kit and BeaverBeadsTM Circulating DNA Kit, respectively. Then real time fluorescence quantitative PCR was performed with mitochondrial encoded genes ND1 and hmito3 as primers and the content of cffmtDNA extracted was compared. Results： The cfmtDNA concentration obtained by one step centrifugation was significantly higher than the other three methods（P〈0.05）. There was no difference among the other three methods（P〈 0.05）. The cfmtDNA concentration from frozen samples was not different from fresh samples（P〈0.05）. The concentration of cfmtDNA amplified with ND1 primers was significantly higher than that with hmito3 primers（P〈0.05）. However, the concentration of cfmtDNA amplified by primers ND1 was significantly correlated with that by hmito3（r= 0.63,P〈0.000 1）. The concentration of cfmtDNA extracted by BeaverBeadsTM Circulating DNA Kit was significantly higher than that by TIANamp Genomic DNA Kit （P〈0.05）. Conclusions： The treatment of follicular fluid samples, the choice of primers and kits influence the quantification of cfmtDNA in follicular fluid. It is recommended to use two step centrifugation to treat follicular fluid samples,magnetic beads method to extract free DNA, and ND1 primers for quantificaion of cfmtDNA.