Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR.
Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR.
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DOI：
10.1371/journal.pone.0164046
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Nussey DH
中科院分区：
文献类型：
--
作者：
Seeker LA;Holland R;Underwood S;Fairlie J;Psifidi A;Ilska JJ;Bagnall A;Whitelaw B;Coffey M;Banos G;Nussey DH
在流行病学、生物医学和生态学研究中，端粒长度越来越多地被用作生物标志物。DNA提取技术已广泛应用于端粒实验，最近基于定量聚合酶链式反应(QPCR)的研究表明，DNA提取方法的选择可能会影响平均相对热释光(RTL)的测量。这种提取方法的影响可能会限制用不同方法提取的历史收集的DNA样本的使用。然而，如果提取方法的影响是系统性的，则特定于提取方法(MS)的校准器可能能够对其进行校正，因为系统效应将以与所有其他样品相同的方式影响校准器样品。在本研究中，我们测试了定量聚合酶链式反应测定荷斯坦公牛和苏伊绵羊血液中的白细胞RTL是否受到DNA提取方法的影响，以及MS校准是否能解释观察到的差异。我们比较了两种硅膜DNA提取试剂盒和一种盐析法。对所有的提取方法进行了优化，以获得足够高质量的DNA用于热释光测定。在这两个物种中，我们发现，当使用相同的校准器进行校准时，基于二氧化硅薄膜的DNA提取方法比非基于薄膜的方法产生的RTL测量更短。然而，当使用MS校准器计算RTL时，这些差异在统计上无法检测到。这种方法产生的RTL测量结果在不同提取方法之间高度相关(r>；0.76)，并且在不同方法提取的相同样品的平板上的变异系数低于10%。我们的结果与以前的发现是一致的，即流行的基于膜的DNA提取方法可能导致比非基于膜的方法更短的RTL测量。然而，我们也证明了这些差异可以通过使用提取方法特定的校准器来解释，这为研究人员提供了一种简单的方法来解释研究中不同DNA提取方法提取的样本RTL测量的差异。
Telomere length (TL) is increasingly being used as a biomarker in epidemiological, biomedical and ecological studies. A wide range of DNA extraction techniques have been used in telomere experiments and recent quantitative PCR (qPCR) based studies suggest that the choice of DNA extraction method may influence average relative TL (RTL) measurements. Such extraction method effects may limit the use of historically collected DNA samples extracted with different methods. However, if extraction method effects are systematic an extraction method specific (MS) calibrator might be able to correct for them, because systematic effects would influence the calibrator sample in the same way as all other samples. In the present study we tested whether leukocyte RTL in blood samples from Holstein Friesian cattle and Soay sheep measured by qPCR was influenced by DNA extraction method and whether MS calibration could account for any observed differences. We compared two silica membrane-based DNA extraction kits and a salting out method. All extraction methods were optimized to yield enough high quality DNA for TL measurement. In both species we found that silica membrane-based DNA extraction methods produced shorter RTL measurements than the non-membrane-based method when calibrated against an identical calibrator. However, these differences were not statistically detectable when a MS calibrator was used to calculate RTL. This approach produced RTL measurements that were highly correlated across extraction methods (r > 0.76) and had coefficients of variation lower than 10% across plates of identical samples extracted by different methods. Our results are consistent with previous findings that popular membrane-based DNA extraction methods may lead to shorter RTL measurements than non-membrane-based methods. However, we also demonstrate that these differences can be accounted for by using an extraction method-specific calibrator, offering researchers a simple means of accounting for differences in RTL measurements from samples extracted by different DNA extraction methods within a study.
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