Telomere length measurements using digital fluorescence microscopy.
Telomere length measurements using digital fluorescence microscopy.
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DOI：
10.1002/(sici)1097-0320(19990801)36:4
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发表时间：
1999-08
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通讯作者：
Steven S. S. Poon-Steven-S.-S.-Poon-144902785;U. Martens;R.K. Ward;P. Lansdorp
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文献类型：
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作者：
Steven S. S. Poon-Steven-S.-S.-Poon-144902785;U. Martens;R.K. Ward;P. Lansdorp
背景染色体的末端（端粒）对于维持染色体稳定性是重要的，并且端粒重复序列的丢失与癌细胞的细胞衰老和基因组不稳定性有关。测量端粒长度的传统方法（Southern分析）需要大量的细胞（>10（5）），并且不能提供单个染色体端粒长度的信息。在这里，我们描述了一个数字图像显微镜系统，用于定量荧光原位杂交（Q-FISH）后的中期细胞中的端粒重复序列的荧光强度的测量。方法用Cy 3标记的肽核酸探针（T（2）AG（3））（n）序列和DNA染料DAPI制备用于显微镜检查的样品。采集Cy 3和DAPI荧光的单独图像，并用专用计算机程序（TFL-TELO）处理。使用该程序，计算每个端粒的积分荧光强度值，其与杂交探针的数量成比例，并呈现给用户。结果我们的方法的间接测试进行模拟以及定义的测试对象。然后在许多实验中分析了人类端粒长度测量的精确度和一致性。发现通过平均小于30个细胞的结果，可以获得端粒长度的良好指示（SD为10-15%）。结论：我们证明，准确和可重复的荧光强度测量可以从Q-FISH图像，提供信息的端粒重复序列的长度在单个染色体从有限数量的细胞。
BACKGROUND The ends of chromosomes (telomeres) are important to maintain chromosome stability, and the loss of telomere repeat sequences has been implicated in cellular senescence and genomic instability of cancer cells. The traditional method for measuring the length of telomeres (Southern analysis) requires a large number of cells (>10(5)) and does not provide information on the telomere length of individual chromosomes. Here, we describe a digital image microscopy system for measurements of the fluorescence intensity derived from telomere repeat sequences in metaphase cells following quantitative fluorescence in situ hybridization (Q-FISH). METHODS Samples are prepared for microscopy using Q-FISH with Cy3 labeled peptide nucleic acid probes specific for (T(2)AG(3))(n) sequences and the DNA dye DAPI. Separate images of Cy3 and DAPI fluorescence are acquired and processed with a dedicated computer program (TFL-TELO). With the program, the integrated fluorescence intensity value for each telomere, which is proportional to the number of hybridized probes, is calculated and presented to the user. RESULTS Indirect tests of our method were performed using simulated as well as defined tests objects. The precision and consistency of human telomere length measurements was then analyzed in a number of experiments. It was found that by averaging the results of less than 30 cells, a good indication of the telomere length (SD of 10-15%) can be obtained. CONCLUSIONS We demonstrate that accurate and repeatable fluorescence intensity measurements can be made from Q-FISH images that provide information on the length of telomere repeats at individual chromosomes from limited number of cells.