Denaturation and Hybridization
Fifteen microliters of hybridization mixture consisting of 0.3 µg/mL Cy3-labeled, telomere-specific PNA probe; 0.1 µg/mL FITC-labeled, chromosome 9-specific Sat III PNA probe; 50% formamide; 10 mM Tris-HCl, pH 7.5; 5% blocking reagent; and 1X Denhart’s solution was applied to each slide and covered under a cover slip.
Before starting the procedure, SciGene’s Hybex microarray incubation system was preheated to 75°C with a water bath insert in position. Fifteen microliters of hybridization mixture was pipetted onto each slide, and the spread area was covered with a cover slip, avoiding the creation of air bubbles.
Then, the slides were inserted into the Hybex slide racks, which were mounted on a hybridization chamber base (Figure 1A). One milliliter of water was pipetted on the absorbent pad situated in the chamber cover to provide adequate moisture during hybridization. The cover of the chamber was sealed to the base by tightening the corner screws (Figure 1B), and a black handle attached. The water bath insert was removed from the preheated Hybex unit, and the assembled chamber was placed into the heating unit, using both chambers to ensure accurate heat control (Figure 1C).
Once the system temperature equilibrated to 75°C (in 8–10 minutes), the slides were incubated for five minutes. Afterward, the hybridization chambers were removed and left at room temperature for 15 minutes. The Hybex unit was speed-cooled by setting the temperature to 30°C and placing a water bath insert into the unit. When 30°C was reached, the hybridization chambers were placed back into the instrument and incubated for three hours. (For DNA probes, hybridization can be performed at 37–42°C for 10 to 16 hours.)
Posthybridization Processing of Slides
Four staining dishes were prewarmed in a water bath set at 42°C, each contained 200 mL of 2X SSC, 1X SSC, 0.5X SSC, and 0.1X SSC. The hybridization chambers were removed from the Hybex incubator, corner screws loosened, covers lifted off, and slide racks removed. The racks were placed into the dish containing 2X SSC at 42°C for five minutes. The processing handle was attached to the slide rack and agitated up and down to release cover slips.
The slide rack was then moved to a second dish containing 1X SSC at 42°C and soaked for 10 minutes. The wash was repeated with 0.5X SSC and 0.1X SSC for ten minutes each at 42°C. Slides were dehydrated in the ethanol series (two minutes each in 70%, 80%, 90%, and 100%) and air-dried. Following the drying of slides, 15 µl of antifade solution with DAPI was added to each slide, and spread area was covered with a cover slip.
The sample slide was analyzed using a Leica DM 4000 epifluorescence microscope equipped with short-arc mercury lamp illumination, a 100X/1.3 N.A. oil immersion neofluotar objective lens, and appropriate band pass filters for Cy3, FITC, and DAPI. Fluorescent images were captured with a charge-coupled device camera.
Figures 2 and 3 demonstrate that the FISH procedure using the Hybex microarray incubation system can be utilized to generate bright and high-quality telomere signals. We also observed that the brightness of telomere signals was uniform across different areas of the slide with low background noise. The improved signal-to-noise ratio increased the sensitivity of the method, allowing for the measurement of short telomeres in cancer cells (data not shown).
To test the reproducibility of the method, we performed three repeat experiments one week apart, each with five samples. The Table shows that the coefficient of variation of these repeat experiments is 9%. The method described in this article shows significant improvement over the method previously used in our laboratory with a conventional hybridization oven, delivering data with high reproducibility.
Rajiv Raja, Ph.D. (firstname.lastname@example.org), is director of genomics applications at SciGene, Zuguo Li, Ph.D., is postdoctoral fellow, and Yun-Ling Zheng, Ph.D., is assistant professor at Lombardi Comprehensive Cancer Center at Georgetown University.