By John A. Double, Michael J. Thompson
John A. Double and Michael J. Thompson have accrued a significantly very important sequence of novel and crucial suggestions for learning telomeres and telomerase. those with no trouble reproducible tools supply state of the art instruments to spot, degree, and research telomeres, to figure out telomerase expression on the RNA point, to figure out telomerase job, and to realize power modifiers of this job. The strategies for assaying telomerase job variety from normal radiological seize assays to nonradioactive tools, from non PCR-based easy methods to recommendations utilizing real-time PCR. Telomeres and Telomerase: tools and Protocols offers the middle array of efficient options wanted at the present time to enhance telomerase inhibitors or diagnostic/prognostic telomerase markers
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Extra info for Telomeres and Telomerase: Methods and Protocols
3A ). 7. Perform global thresholding so that all of the telomere spots are detected; the thresholding level must be sufficiently low to detect all of the spots in their entirety but also sufficiently high to avoid noise detection. Transform the image into a binary image with the Make binary command (Fig. 3A ). Telomere Length Distribution 45 8. Save the binary image of the masks of the telomere spots. 9. Because it is easier, in the following telomere measurement step, to visualize simultaneously the detected binary spots and the chromosome, these additional steps can be performed: a.
36 Pommier and Sabatier 13. Whole chromosome painting probes (WCP; ONCOR or Biosys). 14. Antibody Fab fragments for WCP probe detection: Anti-biotine, Goat + anti-goat/FITC (Vector, Biosys), anti-Digoxigenine/FITC or anti-dig/Mouse+anti-mouse/TRITC (Boehringer). 3. 1. Chromosome Preparations 1. Add colcemid (4 µg/mL) during 1- or 2-hours to actively growing cells to accumulate metaphases. 2. Pellet mitotic cells by centrifuging the culture medium at room temperature for 7 min at 400g. 3. 5 mL of 75 mM KCl; add water for a total volume equal to 10 mL (see Note 3).
Genet. 2, 363 – 412. de Lange, T. (1998) Ending up with the right partner. Nature 392, 753-754. Therkelsen, A. , and Kolvraa, S. (1995) Staining of human telomeres with primed in situ labeling (PRINS). Cytogenet. Cell Genet. 68, 115 –118. Krejci, K. and Koch, J. (1998) Improved detection and comparative sizing of human chromosomal telomeres in situ. Chromosoma 107, 198 – 203. Ijdo, J. , Wells, R. , and Reeders, S. T. (1991) Improved telomere detection using a telomere repeat probe (TTAGGG)n generated by PCR.