Cells were treated with 0.5 μg/ml of Colcemid for 4–7 h before harvest. Chromosomes were fixed and telomere PNA-FISH performed with a 5′-Cy3-OO-(CCCTAA)4-3′ probe (PANAgene) as described 14 (link), 63 (link). For CO-FISH, metaphase spreads were incubated sequentially with 5′-Cy3-OO-(CCCTAA)4-3′ and 5′-FAM-CO-(TTAGGG)4-3′ probes. Images were captured on a Nikon Eclipse 800 microscope and processed with MetaMorph Premier (Molecular Devices).
Protocol detail
Telomere Length Analysis by PNA-FISH
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Corresponding Organization :
Other organizations : Yale University, University of Michigan–Ann Arbor, Baylor College of Medicine, Colorado State University, Shanghai Institutes for Biological Sciences, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences
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Variable analysis
independent variables
- Colcemid concentration (0.5 μg/ml)
- Colcemid treatment duration (4–7 h)
- Chromosome morphology
- Telomere structure/length (measured by PNA-FISH)
- Specific DNA strand orientation (measured by CO-FISH)
- Chromosome fixation protocol
- PNA-FISH probe (5'-Cy3-OO-(CCCTAA)4-3')
- CO-FISH probes (5'-Cy3-OO-(CCCTAA)4-3' and 5'-FAM-CO-(TTAGGG)4-3')
- Microscope and imaging software used (Nikon Eclipse 800 and MetaMorph Premier)
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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