O'Sullivan Jacintha N, Finley Jennifer C, Risques Rosa-Ana, Shen Wen-Tang, Gollahon Katherine A, Rabinovitch Peter S
St Vincent's University Hospital, Elm Park, Dublin, Ireland.
Curr Protoc Cytom. 2005 Aug;Chapter 12:Unit 12.6. doi: 10.1002/0471142956.cy1206s33.
Telomeres are repetitive DNA sequences at the end of each chromosome that provide stability and prevent end-to-end chromosome fusions. In order to understand mechanisms responsible for telomere shortening, it is necessary to develop methods for accurate telomere length measurement that can be applied to archival and fresh tissue and cells. This unit describes in situ-based quantitative fluorescence in situ hybridization (QFISH) protocols using a fluorescence-conjugated telomere probe (peptide nucleic acid, PNA) that stains telomeres proportionally to their length. These protocols can be used on formalin-fixed paraffin-embedded tissue, lightly fixed tissue, cells isolated from tissue, cultured cells, and agar-embedded cells. The basic protocol for QFISH staining is modified to achieve excellent QFISH staining for a variety of cell preparations. Image-analysis techniques to quantitate average telomere lengths from tissues and isolated stained cells are also described.
端粒是位于每条染色体末端的重复DNA序列，可提供稳定性并防止染色体端对端融合。为了了解导致端粒缩短的机制，有必要开发可应用于存档组织、新鲜组织及细胞的准确测量端粒长度的方法。本单元介绍了基于原位的定量荧光原位杂交（QFISH）方案，该方案使用与荧光偶联的端粒探针（肽核酸，PNA），其可按端粒长度比例对端粒进行染色。这些方案可用于福尔马林固定石蜡包埋组织、轻度固定组织、从组织中分离出的细胞、培养细胞以及琼脂包埋细胞。QFISH染色的基本方案经过改良，可对各种细胞制剂实现出色的QFISH染色效果。还介绍了用于定量组织和分离出的染色细胞中端粒平均长度的图像分析技术。
