Aging Aging

68 Valenzuela and Effrosfor cells that have short telomeres. Longer telomeres, because they have moreTTAGGG repeats, give a stronger signal than short telomeres.The term “TRF length” is not synonymous with “telomere length.” The TRFincludes both the telomere repeats and the adjacent subtelomeric region that containsboth repetitive and nonrepetitive sequences .3. We suggest using ladders at both ends of the gel. Use 0.5 µL/lane of ladder labelingreaction mix when using freshly prepared material. If the ladder was preparedat an earlier time, add up to 1 µL/lane, but beware of adding too much lest thesignal be overexposed, making it impossible to quantitate TRF length. If thisproblem occurs, one may either add more (>2 µg/lane) genomic DNA, or a smallerquantity of labeled DNA ladder per lane (but not less than 5 µg/lane).Gel orientation can be marked in a number of ways, such as by cutting away acorner of the gel, loading two lanes with the ladder on one side of gel, or loadingcontrol TRF DNA on one side of gel.4. Note that a 0.5% agarose gel is extremely fragile, and we suggest using a spatulaor the back of a Pyrex dish during all transfers. Gels can be stored for up to 2 d atthis stage, for example, if the probe is not ready. This can be done by placing thegel in a sealed plastic bag with 2 mL of 2× SSC at 4°C, as described in Subheading3.5. If the background radiation is too high, place gel once for 1 h (or twice for 30 mineach) at 48°C in 0.1× SSC. Shortening exposure time may also reduce background.If the problem continues, one should increase the amount of DNA, whichreduces the noise by reducing the background. Adding lower concentrations ofprobe will also help reduce the background.If there is no signal, lengthen exposure time. Verify that probe was properlylabeled. Verify that hybridization solution has no precipitates. If precipitate isseen, it is necessary to prepare fresh hybridization solution.6. The mean TRF length is calculated by integrating signal intensity over TRF distributionon gel as a function of mol wt. Divide a scanned TRF image into a gridin which columns cover the entire length of TRF sample analyzed and there are atleast 30 boxes dividing each column (Fig. 2). The following equation can then beused to calculate the mean TRF length:Mean TRF length = Σ(OD i·L i ) / Σ(OD i )where OD i is the intensity signal and L i is the mol wt at a particular (i) box in thegrid as compared to a size marker from a ladder. Before using above equation forTRF length analysis, background must be subtracted from OD i . For each samplethe background can be calculated by averaging the OD from the top two boxesand bottom two boxes adjacent to the smear. The average background OD is thensubtracted from each box in the grid for that particular sample. Alternatively,some phosphorimagers (Packard, Instant Imager) come included with softwarethat can quantitate the intensities of signals to obtain mean values that are plottedto preassigned values from a ladder.