The Octa Chrome Fish assay simultaneously examines all 24 human chromosomes in a single hybridization on one slide. First, prepare metaphase spreads on the specific eight square slide. Then perform an automated scan of the sample slide to facilitate location of all the meta phases and future reevaluation of all cell images.
Next position, the eight square slide over the Octa chrome device and hybridize the probes. Results obtained can show three different painted whole chromosomes in each square based on the arrangement of chromosome combinations on the RO device. I first had the idea for this RO FISH method when I started to apply fish in human population studies during the early 1990s.
Then I introduced my idea of triple color painting, eight square slide to set the cell at the annual A A CR conference in 1994. The main advantage of this technique over existing methods like classic chromosome bending and modern spectral prototyping is that it can readily analyze all 24 human chromosomes in a larger number of cells. Begin with a preparation of metaphase cells fixed in suspension as a control pipette drop of cells onto a slide and verify appropriate density.
Now dip in eight square OC chrome, slide into pure ethanol for two minutes. In a sequence of alternating squares, distribute five microliters of cell suspension onto the template slide to prevent the cell spreads from interfering with each other. Air dry the samples before spotting adjacent pairs.
Apply 20 microliters of 0.2 micrograms per milliliter of DAPI stain to the center of each half of the slide, and place a cover slip. Now, perform an automated scan of the sample slide to localize the metaphase cells. Use metaverse software to facilitate location of all the meta phases and future reevaluation of all cell images.
View the scanning results manually and delete non metaphase artifacts. First, remove the DPI stain in two XSSC buffer, then dehydrate the samples through a series of ethanol washes. Air dry the slide and then place it on a 37 degree Celsius hop plate for five minutes.
Now equilibrate the chroma probe, multi probe hybridization chamber and the hybridization buffer in a 37 degree Celsius water bath With the label side down. Put the OC chrome device on a 37 degrees Celsius hot plate. Add two microliters of prewarm hybridization solution to each of the eight areas.
Carefully invert and orient the template. Slide over the OC chrome device. Verify that the template slide is aligned to the Octa Chrome device.
Then carefully lower the slide onto the drops of hybridization solution and apply gentle even pressure to spread the hybridization solution to the edges of each of the raised areas on the OC chrome device. Lift at the frosted end of the glass slide and invert so that the slide is underneath the OC chrome device. Place on a 37 degree Celsius hot plate for 10 minutes.
Then transfer it to a 75 degree Celsius hot plate for five minutes. To denature the samples for the hybridization step, transfer to a chroma probe, multi probe hybridization chamber. Replace the lid and float the chamber in the 37 degree Celsius water bath overnight.
Remove the slide carefully from the device and wash in 0.4 excess SC at 72 degrees Celsius for two minutes. Perform the second wash at room temperature for 30 seconds. Proceed to add 20 microliters of DPI to the center of each half of the slide and position a cover slip.
Incubate the samples in the dark for 10 minutes at room temperature before viewing by fluorescence microscopy. The Octa chrome fish assay facilitates simultaneous examination of all 24 human chromosomes using a single hybridization on a single slide. Each square carries three different whole chromosome painting probes directly labeled with different colored fluoro.
Fours examination of a normal metaphase cell in square two of the Octa CHRO system shows that chromosomes 8 21 and 12 are painted red, green, and blue. They can be visualized individually using respective filters or visualized simultaneously through a DAPI Fitzy, Texas Red triple filter. This chromosomes approach has utility in detection of abnormal cells with leukemia specific chromosomal, translocation, and aneuploidy.
For example, T 8 21 is a common chromosomal translocation in acute myeloid leukemia. Trisomy 21 3 copies of chromosome 21 is a common aneuploidy in leukemia After the development. This technique paved the way for scientists in the field of molecular cytogenetics and molecular epidemiology to explore the mostly common chromosomal rearrangements detected in leukemia and the lymphoma, and to examine selective ploidy among all human chromosomes in clinical patients or populations exposed to toxical chemicals.
After watching this video, you should have a good understanding of how to simultaneously examine all 24 human chromosomes in a single hybridization on one slide by the Octo Chrome Fish assay. And we define this as chromosomes.
