摘要
目的综合评估3种游离核酸(cfDNA)提取试剂盒,以筛选出经济可靠的商品试剂盒。方法我们使用了3种不同的试剂盒提取10名健康人血浆中的cfDNA,并使用Agilent 2100生物芯片分析仪对cfDNA片段大小和丰度进行评估。同时,我们还使用微滴式数字PCR(ddPCR)对cfDNA中的核糖核酸酶P蛋白亚基p30(RPP30)、NADH脱氢酶亚基1(ND1)、NADH脱氢酶亚基4(ND4)基因拷贝数进行定量,并利用实时荧光定量PCR(qPCR)检测cfDNA中藤黄节杆菌序列(ALU)ALU115和ALU247的浓度。此外,我们还在混合血浆中掺入50~766 bp的DNA Ladder,然后使用上述3种提取试剂盒对其进行提取,并使用Agilent 2100生物芯片分析仪分析各DNA片段的浓度,以计算不同试剂盒对不同大小的DNA片段的回收率和提取偏好性。结果3种试剂盒从健康人血浆中纯化的100~300 bp cfDNA浓度分别为(5.10±1.99)、(4.64±3.09)和(1.82±1.29)ng/mL(P=0.006)。Kit A所提取的RPP30拷贝数显著高于Kit C(P=0.018),而Kit A和Kit B所提取的ND1和ND4拷贝数也均显著高于Kit C(P=0.001;P=0.001;P=0.001;P=0.001)。此外,Kit A所提取的cfDNA中,ALU 115和ALU 247的浓度也显著高于Kit C(P=0.009;P=0.003)。3种试剂盒对混合血浆中掺入的外源性DNA Ladder的回收率差异有统计学意义(P<0.001),同时Kit B和Kit C对不同大小的DNA片段也存在提取偏好性。结论Kit A性能最佳,Kit B是一种低成本替代方案,其工作流程更简单省时。
Objective To comprehensively evaluate three commercially available cell⁃free DNA(cfDNA)extraction kits and select the economical and reliable ones.Methods Three cfDNA extraction kits were used to puri⁃fy cfDNA from plasma samples of 10 healthy individuals,and the size distribution and the abundance of each DNA fragment in the cfDNA samples were assessed using an Agilent 2100 bioanalyzer.The copy numbers of the ribonucle⁃ase P protein subunit p30(RPP30),the NADH dehydrogenase subunit 1(ND1),and the NADH dehydrogenase subunit 4(ND4)in cfDNA were quantified using droplet digital PCR(ddPCR),and the concentrations of Arthro⁃bacter luteus(ALU)repeats ALU115 and ALU247 in cfDNA were determined using quantitative real time PCR(qPCR).In addition,a DNA Ladder with fragment lengths ranging from 50 bp to 766 bp was spiked into a mixed plasma sample and extracted using the abve three kits mentioned above.The concentrations of the spiked⁃in DNA fragments were determined using an Agilent 2100 bioanalyzer to monitor the extraction efficiency and size bias of the three kits.Results The concentrations of 100~300 bp cfDNA obtained by the three kits from the plasma of 10 healthy individuals were(5.10±1.99),(4.64±3.09)and(1.82±1.29)ng/mL,respectively(P=0.006).We found that the copy number of the RPP30 extracted by Kit A was significantly higher than that by Kit C(P=0.018),and the copy number of the ND1 and the ND4 in cfDNA extracted by both Kit A and Kit B were also significantly higher than those by Kit C(P=0.001;P=0.001;P=0.001;P=0.001).In addition,the concentration of ALU115 and ALU247 in cfDNA extracted by Kit A were significantly higher than those by Kit C(P=0.009;P=0.003).We de⁃tected significant differences in the extraction efficiency of the three kits for the spiked⁃in DNA Ladder(P<0.001).Kit B and Kit C also displayed obvious fragment size bias for DNA recovery.Conclusion Kit A shows the best performance,while Kit B is a low⁃cost alternative with a simpler and faster workflow.
作者
应超
蔡燕宁
郝淑文
王婷
刘沅贺
赵立芳
YING Chao;CAI Yanning;HAO Shuwen;WANG Ting;LIU Yuanhe;ZHAO Lifang(Neurobiology Laboratory,Xuanwu Hospital,Capital Medical University,Beijing 100053,China;Key Laboratory of Neurodegenerative Diseases,Ministry of Education,Beijing 100053,China;National Clinical Research Center for Geriatric Diseases,Beijing 100053,China;不详)
出处
《实用医学杂志》
CAS
北大核心
2023年第12期1556-1563,共8页
The Journal of Practical Medicine
基金
国家重点研发计划(编号:2021YFC2501205)。
关键词
游离DNA
提取试剂盒
核酸提取
Cell⁃free DNA
extraction kit
nucleic acid extraction
