Absolute standards as a useful addition to the avian quantitative PCR telomere assay
Absolute standards as a useful addition to the avian quantitative PCR telomere assay
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DOI：
10.1111/j.1600-048x.2012.05787.x
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发表时间：
2012-11-01
影响因子：
1.7
通讯作者：
Richardson, David S.
中科院分区：
文献类型：
--
作者：
Barrett, Emma L. B.;Boner, Winifred;Richardson, David S.
鸟类种群为研究野外寿命的变化提供了极好的系统，因为个体通常可以在其一生中进行监测。最近的一些研究表明，保护端粒染色体帽的动态（端粒长度和损失率）是生物状态的指示，并可能作为未来寿命的指标。目前，终端限制片段（Terminal Restriction Fragment， TRF）分析和相对定量PCR （relative quantitative PCR, qPCR）技术用于鸟类端粒的测定，但存在局限性。TRF分析是耗时的，而相对qPCR给出了一个研究内的相对值，使得难以在实验之间进行比较。利用首次在人类中开发的方法，使用合成寡聚物端粒（TTAGGG）n和已知长度的正常化基因标准来校准qPCR值，我们描述了一种适用于鸟类qPCR端粒测定的方法，使结果在鸟类内部和鸟类之间具有可比性。我们对塞舌尔莺Acrocephalus sechellensis的绝对qPCR方法与相同样品的相对qPCR测量结果进行了评估。两种方法的端粒估计都显示出端粒长度与年龄相关的下降，并且高度相关（r = 0.99）。绝对qPCR鸟类端粒分析可能被证明是一种有用的手段，估计端粒长度在校准，敏感，有效的方式使用少量的鸟类存档样本。
Bird populations provide excellent systems to investigate variation in longevity in the wild since individuals can often be monitored over their lifetime. A number of recent studies suggest that the dynamics of protective telomere chromosome caps (telomere length and rate of loss) are indicative of biological state and potentially useful as indicators of future longevity. Currently, Terminal Restriction Fragment (TRF) analysis and relative quantitative PCR (qPCR) are used to measure telomeres in birds, but with limitations. TRF analysis is time consuming, while relative qPCR gives a within-study relative value making it difficult to compare across experiments. Utilising an approach first developed in humans of using synthetic oligomer telomeric (TTAGGG)n and normaliser gene standards of known length to calibrate qPCR values, we describe a methodological adaptation to the avian qPCR telomere assay to make results comparable within, and potentially between, bird species. We evaluate this absolute qPCR method in the Seychelles warbler Acrocephalus sechellensis against relative qPCR measurements on the same samples. Telomere estimates from both methods showed an age-related decline in telomere length, and were highly correlated (r = 0.99). Absolute qPCR avian telomere analysis may prove a useful means of estimating telomere lengths in a calibrated, sensitive, and efficient way using small amounts of archived bird sample.