5种血浆游离DNA提取方法的比较

Comparison of five methods of plasma DNA extraction

目的分别应用5种方法进行血浆游离DNA的提取,从而判定与筛选最佳的血浆cfDNA提取方法。方法采集19例健康人血浆标本,针对每一样本取350μl血浆分别应用经1次/2次酚-氯仿-异戊醇抽提法,经1次/2次酚-氯仿-异戊醇抽提配合微量DNA助沉剂法和介孔纳米磁珠法5种方法进行cfDNA提取,应用紫外分光法测定提取cfDNA的含量与纯度,应用实时荧光定量PCR方法以提取血浆cfDNA为模板进行扩增反应,以验证提取效果。结果应用5种方法从19例350μl血浆样本中提取的血浆cfDNA含量分别为(6971.43±1934.40)ng/ml、(2196.86±823.94)ng/ml、(12397.14±4197.37)ng/ml、(5568.29±1618.17)ng/ml和(7458.57±3706.83)ng/ml,A260/A280值分别为0.93±0.07、1.09±0.12、1.15±0.05、1.16±0.12和0.89±0.19,RT-qPCR扩增成功率为100%。结论经1次酚-氯仿-异戊醇配合微量DNA助沉剂法提取量大,纯度高,所提取的血浆cfDNA可以为RT-qPCR提供模板,进行后续实验。

Objective To screen an optimal method of plasma cell-free DNA （cfDNA） extraction among five methods. Methods Plasma samples were collected from 19 healthy persons. 350 μl plasma of each sample was taken to extract the cf DNA by once/twice phenol-chloroform-isoamyl alcohol extraction, once/twice phenol-chloroform-isoamyl alcohol extraction combined with DNA down, and mesoporous nano-beads methods. The concentration and purity of cfDNA was measured by UV spectrophotometry Real-time quantitative PCR （RT-qPCR） was performed using the plasma cfDNA as a template to deter- mine the extraction efficacy. Results The concentrations of plasma cfDNA extracted by the five methods from 19 cases were （6971.43 ± 1934.40） ng/ml, （2196.86 ± 823.94 ） ng/ml, （ 12397. 14 ± 4197.37 ） ng/ml, （5568.29 ±1618. 17） ng/ml, and （7458.57 ± 3706.83 ） ng/ml, and the A260/A280 values were 0.93 20.07, 1.09 20. 12, 1.1520.05, 1.1620.12, 0.89 20. 19, respectively. The RT-qPCR amplification success rate was 100%. Conclusions Once phenol-chloroform-isoamyl alcohol extraction combined with DNAdown method can be used to obtain cf DNA in high concentration and purity, and the extracted plasma cf DNA can provide template for RT-qPCR and subsequent experiments