Giemsa Reverse Banding (RHG)

 

 

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.


 

R Banding by Fluorescence Using Acridine Orange (AO)

 

Principle

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

Background

Acridine orange was originally used to stain untreated chromosomes, both human and mouse. Bobrow et al. and Baserga and Castoldi independently reported the use of acridine orange to obtain a reverse banding pattern of chromosomes. Acridine orange (AO) is a base composition-independent fluorochrome that binds to DNA by intercalation and which gives relatively uniform fluorescence along the length of the chromosome arms. The dye binds very little to non-nucleic acid cell components, but it fluoresces orange-red when bound to single-stranded nucleic acids and yellow-green when bound to double-stranded nucleic acids. Following hot phosphate buffer treatment, R bands are yellow-green, and G/Q bands are orange-red. The major factor that contributes to R banding is the relative GC-richness of the R bands. In many laboratories, RHG methods have been abandoned in favor of a fluorescent R-banding technique (Gustashaw, 1991).

Solutions

  • Phosphate Buffer:
    32 ml of 0.07N Na2HPO4 . 12 H20
    68 mL of 0.07 mol/L KH2PO4
    Adjust pH to 6.5 by adding 0.07N Na2HPO4 . 12 H2O to the solution.
  • Acridine Orange: 0.01%, prepared in the phosphate buffer

Procedure

  1. Prewarm the buffer to 85 degrees C.

  2. Incubate one slide for 10 to 30 minutes in hot phosphate buffer.

  3. Stain with 0.01% acridine orange for 4 to 6 minutes.

  4. Rinse in phosphate buffer (pH 6.5) for 1.5 to 3 minutes.

  5. Mount with the some buffer, without sealing the coverslip.

  6. Examine by fluorescence microscopy using the appropriate filter combination (e.g., excitation: 450-490 nm; suppression: 515 nm).

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.

Verma, RS, Lubs HA. Additional observations on the preparation of R banded human chromosomes with acridine orange. Can J Genet Cytol 18:45-50, 1976.

Verma, RS, Lubs HA. A simple R banding technique. Am J Hum Genet 27:110-117, 1975.