| 摘要: |
| 目的 探讨NanoDrop检测D260/D230值对全基因组关联研究（GWAS）中DNA标本质检的意义。方法 收集1 494例强直性脊柱炎（AS）患者的血液DNA样本，分别用NanoDrop和PicoGreen检测样本浓度。第一阶段，对24例NanoDrop和PicoGreen浓度>50 ng/μL的DNA样本行Omni中华8芯片检测，比较16例芯片成功样本与8例失败样本的D260/D280值及D260/D230值。第二阶段，选取1 122例NanoDrop和PicoGreen检测浓度均大于50 ng/μL DNA样本行管家基因GAPDH的PCR检测，并对PCR反应成功的样本行Omni中华8芯片检测。采用Mann-Whitney U检验比较PCR反应成功与失败DNA样本的D260/D230值，采用受试者工作特征（ROC）曲线评价D260/D230值对PCR结果的鉴别效率。结果 第一阶段中，芯片成功与失败样本的D260/D280值差异无统计学意义（P=0.444），而D260/D230值差异有统计学意义（Z=-3.920，P<0.001）；第二阶段中，PCR成功的DNA样本基因分型检测成功率为100%，PCR成功与失败组的D260/D230值比较差异有统计学意义（Z=-5.983，P<0.01）。D260/D230值预测PCR结果的曲线下面积（AUC）为0.727；最佳诊断点的D260/D230值为0.89；特异度为0.95时的D260/D230值为2.305。结论 在进行GWAS时，浓度及D260/D280值均较好而D260/D230值较低的DNA样本可能含有较多杂质，需联用PCR检测以确保样本质量；D260/D230值≥ 2.305时，样本纯度满足GWAS芯片检测的要求，可省略PCR检测。 |
| 关键词: 全基因组关联研究 D260/D230值 NanoDrop PicoGreen 质量控制 聚合酶链反应 管家基因 |
| DOI：10.16781/j.0258-879x.2017.11.1444 |
| 投稿时间：2017-08-21修订日期：2017-11-06 |
| 基金项目:国家重点基础研究规划(973计划),编号2014CB541800 |
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| Significance of D260/D230 ratio of NanoDrop detection in quality assay of DNA in genome wide association study |
| GUO Qian△,WANG Geng△,YIN Jian,LI Meng-meng,HUANG Shao-lan,JIANG Lei,XU Hu-ji* |
(Department of Rheumatology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China
△Co-first authors.
* Corresponding author) |
| Abstract: |
| Objective To investigate the significance of D260/D230 ratio of NanoDrop detection in quality assay of DNA in genome wide association study (GWAS). Methods Blood samples from 1 494 patients with ankylosing spondylitis (AS) were collected and the DNA was extracted. The concentrations of DNA samples were detected by NanoDrop and PicoGreen methods. In first stage, 24 DNA samples with concentrations>50 ng/μL were detected by Omni ZhongHua-8 microarray. Among the samples, 16 cases of chip reaction were successful and 8 were failure, and then the ratios of D260/D280 and D260/D230 were compared between the two groups. In second stage, 1 122 DNA samples with a concentration greater than 50 ng/μL according to NanoDrop and PicoGreen tests were selected for PCR detection of house-keeping genes. Samples with successful PCR reaction were detected by Human Omni ZhongHua-8 microarray. The 1 122 samples are divided into two groups according to the results of PCR (successful and failure groups). Mann-Whitney U test was used to compare the ratio of D260/D230 between the two groups, and receiver operating characteristic (ROC) curve was used to evaluate the predictive efficiency of D260/D230 value in PCR results. Results In the first stage, there were no significant differences in D260/D230 values between DNA samples with successful chip reaction and failure chip reaction (P=0.444), while the D260/D230 value of the fomer samples was significantly lower than that of the latter (Z=-3.920, P<0.001). In the second stage, the success rate of genotyping of DNA samples with positive PCR results was 100%. There were significant differences in D260/D230 values between the DNA samples with positive and negative PCR results (Z=-5.983, P<0.01. The area under curve of D260/D230value predicting the PCR results was 0.727; the D260/D230 values of the best diagnostic point and the point of specificity 95% were 0.89 and 2.305, respectively. Conclusion In GWAS, when DNA sample has better concentration and D260/D280 value and has lower D260/D230 value, PCR test should be performed to ensure the quality of the samples; when D260/D230 value is higher than 2.305, the samples are pure enough for microarray detection and the PCR detection can be omitted. |
| Key words: genome wide association study D260/D230value NanoDrop PicoGreen quality control polymerase chain reaction housekeeping gene |
