抗凝血离心沉淀红细胞中残留基因组DNA的质量分析  

Quality Analysis of Residual Genomic DNA in Red Blood Cells Precipitated by Anticoagulation Blood Centrifugation

作　　者：张鑫 肖宇 钱开宇 夏彩霞[2] 朱源 ZHANG Xin;XIAO Yu;QIAN Kaiyu;XIA Caixia;ZHU Yuan(Department of Biological Repositories,Zhongnan Hospital of Wuhan University,Wuhan 430071,China;President's Office,Zhongnan Hospital of Wuhan University,Wuhan 430071,China)

机构地区：[1]武汉大学中南医院生物样本库,武汉430071 [2]武汉大学中南医院院长办公室,武汉430071

出　　处：《转化医学杂志》2023年第2期80-85,共6页Translational Medicine Journal

基　　金：国家卫生健康委员会疑难病症诊治能力提升工程项目(ZLYNXM202006)。

摘　　要：目的探究抗凝血离心后红细胞样本中基因组DNA(gDNA)的质量及不同处理方式对gDNA产量的影响。方法血液样本均来自于武汉大学中南医院生物样本库,采用试剂盒分离柱纯化的方式提取冻存4年及未冻存的抗凝血红细胞样本中gDNA,使用Nanodrop One超微量分光光度计、Qubit 4荧光计和Agilent 4200 TapeStation生物分析仪分别对gDNA样本进行检测,从浓度、纯度、完整性进行样本质量评估。结果从冻存4年的红细胞样本中可提取得到纯度和完整性较高的gDNA,50例250μL冻存4年红细胞样本的gDNA产量中位数为8.80μg,A260/A280比值为1.92±0.08,A260/A230比值为2.23±1.37。与不做处理的未冻存红细胞样本比较,利用红细胞裂解液处理未冻存红细胞样本留取白细胞提取gDNA会使产量降低(P<0.01),而gDNA纯度没有明显差异。未冻存与冻存4年的红细胞样本gDNA产量比较差异无统计学意义(P>0.05)。未冻存白膜层样本gDNA产量明显高于未冻存红细胞样本(P<0.01)。结论抗凝血离心后去除血浆和白膜层的剩余红细胞样本中,仍有少量白细胞残留,选择合适的方法可提取得到纯度较高的gDNA,生物样本库存储红细胞样本能使血液样本得到更充分利用。Objective To explore the quality of genomic DNA(gDNA)in red blood cell samples after anticoagulant centrifugation and the effects of different treatments on gDNA production.Methods Blood samples were collected from the Biological Repositories of Zhongnan Hospital of Wuhan University.gDNA was extracted from anticoagulant red blood cell samples frozen for 4 years and those not frozen for 4 years by kit separation column purification.Nanodrop One ultramicrospectrophotometer,Qubit 4 fluorimeter and Agilent 4200 TapeStation bioanalyzer were used to detect gDNA samples,and sample quality was evaluated in terms of concentration,purity and integrity.Results gDNA with high purity and integrity was extracted from the frozen red blood cell samples for 4 years.The median gDNA yield of 50250μL frozen red blood cell samples for 4 years was 8.80μg,the ratio of A260/A280 was 1.92±0.08,and the ratio of A260/A230 was 2.23±1.37.Compared with unfrozen red blood cell samples without treatment,the yield of unfrozen red blood cell samples treated with red blood cell lysate and leukocyte extraction for gDNA extraction decreased(P<0.01),but the purity of gDNA had no significant difference.There was no significant difference in gDNA production between unfrozen and frozen red blood cell samples for 4 years(P>0.05).The gDNA yield of unfrozen white membrane samples was significantly higher than that of unfrozen red blood cell samples(P<0.01).Conclusion After anticoagulant centrifugation,there are still a small number of white blood cells in the remaining red blood cell samples after the removal of plasma and white membrane layer.The gDNA with high purity can be extracted by selecting appropriate methods.The storage of red blood cell samples in the biosample library can ensure full utilization of the blood samples.

关 键 词：血液样本 红细胞 基因组DNA DNA提取 分光光度法 电泳法 生物样本库 质量控制 

分 类 号：R440[医药卫生—诊断学]