Reproducibility of telomere length assessment: an international collaborative study
Reproducibility of telomere length assessment: an international collaborative study
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DOI：
10.1093/ije/dyu191
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发表时间：
2015-10-01
影响因子：
7.7
通讯作者：
von Zglinicki, Thomas
中科院分区：
文献类型：
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作者：
Martin-Ruiz, Carmen M.;Baird, Duncan;von Zglinicki, Thomas
背景：端粒长度可能是衰老、发病率和死亡率的生物标志物。由于缺乏广泛适用的参考范围，以及目前实验室内和实验室间测量重复性极限的不确定性，它的应用受到了阻碍。方法：我们发起了一项国际合作的端粒长度评估研究：10个不同的实验室，采用3种不同的技术[Southern blotting，单端粒长度分析(STELA)和实时定量聚合酶链式反应(QPCR)]，每轮对10个人DNA样本进行两轮全盲测量，以实现对实验室和技术之间和批间差异的公正评估。结果：不同实验室的绝对结果差异很大，因此不能直接进行比较。但相对端粒长度的排名高度相关(相关系数为0.63-0.99)。Southern blotting和qPCR的技术内相关性相似，但强于技术间相关性。然而，Southern杂交和STELA的实验室间变异系数(CVs)平均约为10%，qPCR的平均变异系数超过20%。这种差异被qPCR方法更高的动态范围所补偿，如z评分后的等方差所示。每个实验室的技术差异，以批内和批间CV的中位数衡量，范围从1.4%到9.5%，实验室之间的差异仅略有显著意义(P=0.06)。凝胶技术和聚合酶链式反应技术在准确性上没有差异。结论：实验室内和实验室间的技术差异严重限制了数据汇集的有效性，并排除了实验室之间参考范围的共享。我们建议建立一套通用的物理端粒长度标准，以提高实验室之间端粒长度估计的可比性。
Background: Telomere length is a putative biomarker of ageing, morbidity and mortality. Its application is hampered by lack of widely applicable reference ranges and uncertainty regarding the present limits of measurement reproducibility within and between laboratories.Methods: We instigated an international collaborative study of telomere length assessment: 10 different laboratories, employing 3 different techniques [Southern blotting, single telomere length analysis (STELA) and real-time quantitative PCR (qPCR)] performed two rounds of fully blinded measurements on 10 human DNA samples per round to enable unbiased assessment of intra- and inter-batch variation between laboratories and techniques.Results: Absolute results from different laboratories differed widely and could thus not be compared directly, but rankings of relative telomere lengths were highly correlated (correlation coefficients of 0.63-0.99). Intra-technique correlations were similar for Southern blotting and qPCR and were stronger than inter-technique ones. However, inter-laboratory coefficients of variation (CVs) averaged about 10% for Southern blotting and STELA and more than 20% for qPCR. This difference was compensated for by a higher dynamic range for the qPCR method as shown by equal variance after z-scoring. Technical variation per laboratory, measured as median of intra- and inter-batch CVs, ranged from 1.4% to 9.5%, with differences between laboratories only marginally significant (P = 0.06). Gel-based and PCR-based techniques were not different in accuracy.Conclusions: Intra- and inter-laboratory technical variation severely limits the usefulness of data pooling and excludes sharing of reference ranges between laboratories. We propose to establish a common set of physical telomere length standards to improve comparability of telomere length estimates between laboratories.