Poon S S, Martens U M, Ward R K, Lansdorp P M
Terry Fox Laboratory for Hematology/Oncology, British Columbia Cancer Research Centre, Vancouver, British Columbia, Canada.
Cytometry. 1999 Aug 1;36(4):267-78. doi: 10.1002/(sici)1097-0320(19990801)36:4<267::aid-cyto1>3.0.co;2-o.
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).
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.
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.
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.
染色体末端（端粒）对于维持染色体稳定性至关重要，端粒重复序列的丢失与细胞衰老及癌细胞的基因组不稳定性有关。传统的测量端粒长度的方法（Southern分析）需要大量细胞（>10⁵），且无法提供单个染色体端粒长度的信息。在此，我们描述了一种数字图像显微镜系统，用于在定量荧光原位杂交（Q-FISH）后测量中期细胞中端粒重复序列的荧光强度。
使用对（T₂AG₃）ₙ序列特异的Cy3标记肽核酸探针和DNA染料DAPI，通过Q-FISH制备用于显微镜观察的样本。采集Cy3和DAPI荧光的单独图像，并使用专用计算机程序（TFL-TELO）进行处理。通过该程序，计算每个端粒的积分荧光强度值，该值与杂交探针的数量成正比，并呈现给用户。
使用模拟以及确定的测试对象对我们的方法进行了间接测试。然后在多个实验中分析了人类端粒长度测量的精度和一致性。发现通过对少于30个细胞的结果进行平均，可以获得端粒长度的良好指示（标准差为10 - 15%）。
我们证明，可以从Q-FISH图像进行准确且可重复的荧光强度测量，这些图像可提供来自有限数量细胞的单个染色体端粒重复序列长度的信息。
