Principle

R-banding methods are useful for analyzing deletions or translocations that involve the telomeres of chromosomes.

Background Reverse banding using heat and Giemsa (RHG) was first described by Dutrillaux and Lejeune. This technique involves the incubation of slides in hot phospate bufer with subsequent Giemsa staining. The resulting chromosome pattern shows darkly stained R bands and pale G bands. R bands are GC-rich and the AT-rich regions are selectively, or more readily, denatured by heat, but the GC-rich regions remain intact. This is consistent with the fact that GC-specific fluorochromes also produce a reverse chromosome banding pattern. In many laboratories, RHG methods have been abandoned in favor of a fluorescent R-banding technique (Gustashaw, 1991).

Solutions

• Buffer:
  10.0 mL Earle's balanced salt solution IEBSS) 10, (GIBCO)
  0.1 mL 7.5% Sodium bicarbonate (GIBCO)
  89.9 mL distilled water
  Place buffer in water bath, and heat to 88 degrees to 89 degrees C.
  
• tap water
  
• 2% Giemsa in distilled water
  

Procedure

1. Incubate slides in hot EBSS for 10 to 15 minutes. (Fresh slides require 1 to 2 hours. One-day-old slides require 25 minutes. One-week-old slides require 7 minutes. In general, older slides require less time.)

2. Cool quickly in tap water. Do not dry.0.

3. Stain in 2% Giemsa for 10 to 20 minutes.

4. Rinse in xylene.

5. Rinse in tap water. Air dry.

References

Gustashaw, KM. Chromosome Stains. In The ACT Cytogenetics Laboratory Manual, Second Edition, edited by M. J. Barch. The Association of Cytogenetic Technologists, Raven Press, Ltd., New York, 1991.