Quantitative fluorescence in situ hybridization (QFISH) of telomere lengths in tissue and cells.
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.
O'Sullivan
Jacintha N
JN
St Vincent's University Hospital, Elm Park, Dublin, Ireland.
Finley
Jennifer C
JC
Risques
Rosa-Ana
RA
Shen
Wen-Tang
WT
Gollahon
Katherine A
KA
Rabinovitch
Peter S
PS
D000818:Animals; D002477:Cells / Q000166:cytology / Q000648:ultrastructure; D002875:Chromosomes / Q000502:physiology / Q000648:ultrastructure; D005557:Formaldehyde; D007091:Image Processing, Computer-Assisted; D017403:In Situ Hybridization / Q000379:methods*; D013050:Spectrometry, Fluorescence / Q000379:methods; D016615:Telomere / Q000648:ultrastructure*
D005557:Formaldehyde