Different methods of telomere length measurement are used to identify patients with telomeropathies. In our lab, we established four different methods for telomere length measurement, terminal restriction fragment (TRF) analysis by Southern blot analysis, quantitative PCR (qPCR), quantitative telomere/centromere fluorescence in situ hybridization (T/C-FISH) and fluorescence in situ hybridization combined with flow cytometry (FlowFISH). The methods each have distinct properties and apart from this-according to our experience and data-may have an impact on the individual result. In this study, we therefore compared and validated these methods measuring 154 healthy individuals of different age groups (newborn-81 years). A linear decline was found for every method (Southern blotting 64 bp per year; qPCR 31 bp per year; T/C-FISH 36 bp per year; FlowFISH 50 bp per year). With the equation of the regression line the values of each method (T/S ratio, T/C value, RTL value) can be expressed in absolute kb. All methods showed acceptable accuracy. The analysis indicated good agreement between all methods, with the best agreement between T/C-FISH and FlowFISH. Here, FlowFISH was the most precise, accurate, and reproducible method compared to the other methods. Based on our data, we emphasize the influence of expertise and experience that is required to produce robust and reliable telomere length analyses. Furthermore, we want to provide the scientific community working in diagnostics and research with data-funded advice on how to choose the appropriate method to safely discriminate between natural variability and pathological telomere shortening in individual cases.

译文

不同的端粒长度测量方法用于识别端粒病患者。在我们的实验室中,我们建立了四种不同的端粒长度测量方法,通过Southern印迹分析,定量PCR (qPCR),端粒/着丝粒荧光原位杂交 (T/C-FISH) 和荧光原位杂交结合流式细胞仪 (FlowFISH) 分析末端限制性片段 (TRF)。根据我们的经验和数据,每种方法都具有不同的属性,除此之外,可能会对单个结果产生影响。因此,在这项研究中,我们比较并验证了这些方法,这些方法测量了154个不同年龄组 (新生儿-81岁) 的健康个体。每种方法均呈线性下降 (每年Southern印迹64 bp; 每年qPCR 31 bp; 每年T/C-FISH 36 bp; 每年FlowFISH 50 bp)。利用回归线的方程,每种方法的值 (T/S比,T/C值,RTL值) 都可以用绝对kb表示。所有方法均显示出可接受的准确性。分析表明所有方法之间具有良好的一致性,T/C-鱼和FlowFISH之间具有最佳的一致性。与其他方法相比,FlowFISH是最精确,准确和可重现的方法。根据我们的数据,我们强调产生可靠而可靠的端粒长度分析所需的专业知识和经验的影响。此外,我们希望为从事诊断和研究的科学界提供数据资助的建议,以了解如何选择适当的方法来安全地区分个别情况下的自然变异性和病理性端粒缩短。

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