Principles Of Qfish

As stated above, Q-FISH requires the use of fluorescently labeled telomeric PNA probes. The most commonly used probe is Cy3-labeled (CCCTAA)3. Following hybridization of this probe with the metaphase chromosome preparations, high stringency washes are performed to eliminate background fluorescence that may interfere with the quantitation of telomere fluorescence. Separate images of metaphase chromosomes and telomeres are then acquired using appropriate image-acquisition software packages and the resulting files can be used for the analysis of telomere fluorescence (Fig. 1). The most common software for the analysis of telomere fluorescence is TFL-TELO developed by Lansdorp's group[10] and this software is available free of charge. The software simply combines images of chromosomes and telomeres, and generates a table with the values of telomere fluorescence for each chromosome, i.e., four telomeres/ chromosome. In this software each telomere is assigned a different color and, also, p-arm and q-arm telomeres can easily be distinguished. The final product is the set of data containing 184 individual telomere fluorescence values (46 chromosomes x 4 telomeres). The files generated by TFL-TELO can be exported to any standard software for statistical analysis. Software packages other than TFL-TELO may be available for Q-FISH on a commercial basis.

To ensure reproducibility of measurements by Q-FISH, it is important to minimize sources of variation associated

Fig. 1 A human metaphase cell. The top panel shows two-color images in which chromosomes are stained in blue (DAPI) and telomeres in red (Cy3 telomeric probe). The middle and bottom images are separate images of chromosomes and telomeres. (View this art in color at www.dekker.com.)

Fig. 1 A human metaphase cell. The top panel shows two-color images in which chromosomes are stained in blue (DAPI) and telomeres in red (Cy3 telomeric probe). The middle and bottom images are separate images of chromosomes and telomeres. (View this art in color at www.dekker.com.)

with digital fluorescence microscopy. The first potential source of variation is the length of exposure time required to acquire optimal images of telomeres. Software packages used for image acquisition usually have two options: auto-exposure and manual exposure. In the case of autoexposure, the exposure time is determined automatically by the image-acquisition software based on the signal strength. In the case of manual exposure desired fixed-time exposure may be selected. Our experience is that if TFL-TELO is used for telomere fluorescence analysis then the appropriate fixed-time exposure selected on the basis of trial and error should be used in all experiments. The second source of variation is associated with variability in microscope fluorescence lamp intensity. To avoid inaccuracies due to lamp intensity variations appropriate internal controls must be used. One option is to use fluorescence beads of defined size (i.e., 1 mm) and acquire images of these beads each time a new sample is analyzed. The values of bead fluorescence will then be used to correct the telomere fluorescence intensities of the samples under investigation. In the original Q-FISH protocol another calibration component was used, plas-mids containing a defined number of telomeric sequence repeats.[8] This approach allowed for the direct conversion of telomere fluorescence into units of DNA length. Another appropriate internal control is the use of cell lines with known telomere length. In this case multiple images of metaphase chromosomes and telomeres from two cell lines with differing telomere lengths are acquired each time a new sample is analyzed, and these values are then used to generate two telomere fluorescence reference points.[11] The telomere fluorescence of the samples under investigation is then expressed relative to these two reference points.

Numerous studies demonstrated the value of Q-FISH. For example, Q-FISH was instrumental in demonstrating telomere dysfunction in mice lacking the RNA component of telomerase. These mice showed progressive telomere shortening which could only be detected by Q-FISH.[12] In addition, Q-FISH was instrumental in demonstrating 1) the heterogeneity of telomere length in individual human and mouse chromosomes;[8,13] and 2) that the shortest telomeres in the cell, not the average telomere length, drive genomic instability due to telomere dysfunction.[7] Finally, the Q-FISH procedure was recently adapted for telomere fluorescence analysis by flow cytometry and this new protocol was named flow-FISH.[14] Flow-FISH analyses the average telomere fluorescence in interphase cells and the advantage of this technique is that it can identify the average telomere length in a large number of cells with higher accuracy than Southern blot. In addition, flow-FISH is useful in identifying subpopulations of cells with differing telo-mere lengths, i.e., different subsets of peripheral blood lymphocytes. The most recent modification of flow-FISH is the use of multicolor flow-FISH in which different subsets of lymphocytes are detected by appropriate antibodies which must be attached to fluorochromes different from fluorochromes attached to the telomeric probe.[15] It is important to stress that Southern blot cannot reliably identify subpopulations of cells with differing telomere lengths in a given cell population. Although Q-FISH theoretically can identify subpopulations of cells with differing telomere lengths in a cell population of interest, in reality this technique is limited for this purpose because it relies on the information from only 10 to 15 cells. In contrast, flow-FISH can analyze thousands of cells in a short period of time.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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