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首页> 美国卫生研究院文献>The Journal of Molecular Diagnostics : JMD >Optimization of a Relative Telomere Length Assay by Monochromatic Multiplex Real-Time Quantitative PCR on the LightCycler 480
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Optimization of a Relative Telomere Length Assay by Monochromatic Multiplex Real-Time Quantitative PCR on the LightCycler 480

机译：在LightCycler 480上通过单色多重实时定量PCR优化相对端粒长度测定

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摘要

Telomere length (TL) measurement is central to many biomedical research, population, and epidemiology studies, with promising potential as a clinical tool. Various assays are used to determine TL, depending on the type and size of the sample. We describe the detailed optimization of a monochromatic multiplex real-time quantitative PCR (MMqPCR) assay for relative TL using the LightCycler 480. MMqPCR was initially developed using a different instrument with many separate reagents. Differences in instrument performance, reagents, and workflow required substantial optimization for the assay to be compatible with the LightCycler 480. We optimized the chemistry of the assay using a purchased one-component reaction mix and herein describe sources of variability and quality control relevant to the MMqPCR TL assay on any instrument. Finally, the assay was validated against other TL assays, such as terminal restriction fragment, Southern blot, and flow fluorescent in situ hybridization. The correlations obtained between data from MMqPCR and these assays (R2 = 0.88 and 0.81) were comparable to those seen with the monoplex version (R2 = 0.85 and 0.82) when the same samples were assayed. The intrarun and interrun CV ranged from 4.2% to 6.2% and 3.2% to 4.9%, respectively. We describe a protocol for measuring TL on the LightCycler platform that provides a robust high-throughput method applicable to clinical diagnostics or large-scale studies of archived specimens.
机译：端粒长度（TL）的测量是许多生物医学研究，人群和流行病学研究的核心，具有作为临床工具的潜力。根据样品的类型和大小，可以使用各种测定方法来确定TL。我们描述了使用LightCycler 480对相对TL进行单色多重实时定量PCR（MMqPCR）测定的详细优化。MMqPCR最初是使用具有许多单独试剂的不同仪器开发的。仪器性能，试剂和工作流程的差异要求对分析进行实质性优化，以使其与LightCycler 480兼容。我们使用购买的单组分反应混合物优化了分析的化学性质，在此描述了与分析仪相关的变异性和质量控制来源在任何仪器上进行MMqPCR TL测定。最后，针对其他TL检测方法（例如末端限制性酶切片段，Southern印迹和流式荧光原位杂交）对检测方法进行了验证。 MMqPCR数据与这些测定（R 2 = 0.88和0.81）之间的相关性与单倍体版本（R 2 = 0.85和0.82）所观察到的相关性可比分析相同的样品。批内和批间CV分别为4.2％至6.2％和3.2％至4.9％。我们描述了一种在LightCycler平台上测量TL的协议，该协议提供了适用于临床诊断或归档标本的大规模研究的可靠的高通量方法。

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