核酸提取效率存在高度的样本间差异，因此需要使用内标对照来基于qPCR准确测量血浆游离DNA水平。
High Interspecimen Variability in Nucleic Acid Extraction Efficiency Necessitates the Use of Spike-In Control for Accurate qPCR-based Measurement of Plasma Cell-Free DNA Levels.
作者信息
O'Connell Grant C, Chantler Paul D, Barr Taura L
机构信息
Center for Basic and Translational Stroke Research, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia.
Department of Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, West Virginia.
出版信息
Lab Med. 2017 Nov 8;48(4):332-338. doi: 10.1093/labmed/lmx043.
OBJECTIVE
To assess the interspecimen variability associated with plasma DNA extraction in order to provide insight regarding the necessity to use an exogenous spike-in control when measuring cell-free DNA (cfDNA) levels using quantitative polymerase chain reaction (qPCR).
METHODS
Plasma specimens were obtained from 8 healthy individuals, 20 patients with cardiovascular disease risk factors, and 54 patients diagnosed with acute stroke. Specimens were spiked with an exogenous oligonucleotide fragment, and total DNA was extracted via automated solid phase anion exchange. We determined recovery of the exogenous fragment via qPCR and used this information to calculate DNA extraction efficiency.
RESULTS
Plasma DNA extraction efficiencies varied dramatically between specimens, ranging from 22.9% to 88.1%, with a coefficient of variance of 28.9%. No significant differences in DNA extraction efficiencies were observed between patient populations.
CONCLUSIONS
We strongly recommend the use of an exogenous spike-in control to account for variance in plasma DNA extraction efficiency when assessing cell free DNA (cfDNA) levels by qPCR in future biomarker investigations.
目的
评估与血浆DNA提取相关的样本间变异性，以便为在使用定量聚合酶链反应（qPCR）测量游离DNA（cfDNA）水平时使用外源性掺入对照的必要性提供见解。
方法
从8名健康个体、20名有心血管疾病风险因素的患者和54名诊断为急性中风的患者中获取血浆样本。样本用外源性寡核苷酸片段掺入，通过自动固相阴离子交换提取总DNA。我们通过qPCR确定外源性片段的回收率，并利用此信息计算DNA提取效率。
结果
样本间血浆DNA提取效率差异很大，范围从22.9%到88.1%，变异系数为28.9%。在不同患者群体之间未观察到DNA提取效率的显著差异。
结论
我们强烈建议在未来的生物标志物研究中，当通过qPCR评估游离DNA（cfDNA）水平时，使用外源性掺入对照来考虑血浆DNA提取效率的差异。
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