T-banding is used to stain the telomeric regions of chromosomes for cytogenetic analysis.
Telomeric (or terminal) banding was first reported by Dutrillaux, who used two types of controlled thermal denaturation followed by staining with either Giemsa or acridine orange. The T bands apparently represent a subset of the R bands because they are smaller that the corresponding R bands and are more strictly telomeric. (Gustashaw, 1991).
T Banding by Thermal Denaturation: Method 1
- Bring 94 mL of distilled water and 3 mL of phosphate buffer (pH 6.7) to 87 degrees C in a Coplin jar.
- Add 3 mL of Giemsa stain.
- Add slides to jar; stain for 5 to 30 minutes.
- Rinse in distilled water, air dry, and examine.
For Fluorescent Observation
- Destain, rehydrate through a series of alcohols, rinse in distilled water.
- Stain in acridine orange (5mg/100mL) for 20 minutes.
- Rinse in phosphate buffer, mount, and examine with a fluorescence microscope (excitation: 450-490 nm; suppression: 515 nm).
Standard culture methods are appropriate. Slides should be aged for a few days prior to staining. With Giemsa (steps 1-4), the chromosomes are pale, unbanded, and difficult to see. With acridine orange staining for various lengths of time, the chromosomes appear as follows: 45 minutes: green at telomeres, otherwise orange; 15 to 20 minutes: green at telomeres, orange areas are less intense; 30 minutes or more: orange color is gone, intercalating R bands appear.
T Banding by Thermal Denaturation: Method 2
- Bring a Coplin jar containing Earle's BSS, PBS, or phosphate buffer to 87 degrees C. The pH must be adjusted to 5.1.
- Stain with Giemsa or acridine orange as in Method 1, steps 2 to 7.
Dutrillaux B. Nouveau systeme de marquage chromosomique: Les bands T. Chromosoma 41:395-402, 1973.
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.