Throughout the history of clinical cytogenetics, G-banding has served as the "gold standard" for detecting total chromosomal abnormalities in clinical samples, from simple quantitative alterations to identifying complex structural rearrangements. 'Marker chromosomes' or 'derived chromosomes' are those abnormalities that cannot be identified and recognized by G-banding. This is usually due to the complexity of the rearrangement resulting in a lack of continuous recognizable banding. Various multicolor fluorescence in situ hybridization (FISH) chromosome coating techniques [1, 2] have emerged that are capable of identifying all marker chromosomes, but the goal of applying these techniques to rapidly identify complex chromosomal rearrangements still needs to be meticulously designed.
Operation method
Spectral chromosome karyotyping
Materials and Instruments
AppliedSpectralImaging ( MigdalHaemek Israel) Kits Tissue Samples Move I. Probes Commercial probes that have been labeled are dissolved in hybridization solution in a mixed form. 1. Aseptically in an ultra-clean bench for tissue culture, in a sterile petri dish, the tissue is chopped into fine pieces with a razor blade. 2. Transfer the finely chopped pieces of tissue into a tissue culture flask with sufficient medium to cover the bottom of the flask placed on a flat surface (usually 10~15 mL) with ventilation holes. 3. Place the flasks in a CO2 incubator and incubate for 24 hours. 4. Observe the growth status under light microscope. 5. When the required growth state or incubation time is reached, add colchicine to the culture flask to a final concentration of l(ug/mL, and continue to incubate in a CO2 incubator for 2~3 h (see Note 1). 6. Transfer the medium to a 15 mL conical-bottomed centrifuge tube along with the suspended tissue or cells (see Note 2). For non-adherent cells, continue with Step 10. 7. Add IOmL of citrate solution to the culture flask, rinse the adherent cells, and discard the citrate solution. 8. Add 10mL of 1 X trypsin to the culture flask. Observe the adherent cells detach from the wall of the culture flask, blow gently, blow the cells off the flask wall, and transfer to a 15 mL conical centrifuge tube. 9. Add 5 mL of freshly prepared medium (see Note 3) to the 15 mL conical centrifuge tube containing the adherent cells. 10. Centrifuge at 1500 g for 10 min to collect cells. 11. Pour off or aspirate the supernatant, leaving only 0.5~1mL of solution. Tap the bottom of the tube gently with a finger to break up the cell mass. 12. Add pre-warmed 0.075mol/L KCl solution, starting with 1mL drop by drop, mixing well between drops (see Note 4); then add the remaining 14mL and mix gently by inverting the mixture; place at 37°C for 10min. 13. Add 5 drops of 3:1 methanol:glacial acetic acid and mix well (see Note 5). 14. Centrifuge at 1500 g for 10 min. 15. Pour off or aspirate the supernatant and break up the cell mass (see Note 6). Add 15 mL of freshly prepared Methanol:Glacial Acetic Acid (3:1) Fixative, starting with ImL as described above and mixing well between drops; then add the remaining 14 mL of Fixative and mix by gently inverting. 16.1500 g, centrifuge for .10 min. 17. Repeat the fixation in step 15. However, the initial 1mL does not need to be added dropwise. Centrifuge and repeat fixation 3 times. 18. The cell suspension can be stored at -20°C in the same state as the cell mass in the fixative. 19.For chromosome specimen preparation, pour off the fixative, resuspend the cell mass, and add an appropriate amount of fresh fixative to make the suspension mildly turbid. 20. Using a 200ul gun, aspirate 100ul of suspension and drop onto a clean slide (see Note 7), allow to dry at room temperature and store the slides. Allow the drops to age naturally for at least 2d or overnight at 37°C. 1. Chromosome specimens should be aged naturally for at least 2d or overnight at 37°C before use. 2. Chromosome specimens are dehydrated in a series of ethanol (70%, 80% and 95%) for 5 min and dried. 3. Add 15-20 uL of 10% pepsin storage solution (see Note 8) to 50 mL of pre-warmed O.Olmol/L HCl, and incubate the chromosome specimens for 5 min at 37°C in a staining vat (see Note 9). At the same time, prepare a vat of 70% formamide/2 X SSC solution and preheat in a 75°C water bath. 4. Remove specimens and rinse in 1 X PBS for 5 min at room temperature. 5. Incubate in 1 X PBS/MgCl2 for 5 min at room temperature. 6. Add 1.5 mL of 37% formaldehyde to 50 mL of l X PBS/MgCl2 and incubate the specimen in this solution for 10 min at room temperature (see Note 11). 7. Rinse for 5 min in 1 X PBS at room temperature. 8. Perform another series of ethanol dehydration as described in step 2. 9. After drying the slices, check that the temperature of the formamide solution reaches 75°C (see Note 11); denature the chromosome specimens in the formamide solution at 75°C for 2 min. 10. Quickly transfer the specimens to ice-cold 70% ethanol and dehydrate them in a series of ethanol baths (see Note 12) and allow to dry. 1. For each specimen, 10uL of probe is required for a 22 mm X 22 mm coverslip; if the area is larger than this, increase the amount of probe used. Take the appropriate amount of probe into an Eppendorf tube. 2. Heat denature the probe for 10 min at 75°C in a water bath or PCR instrument. 3. Pre-denature the probe in a 37°C water bath for 1h. 1. Remove the pre-duplicated probe from 37°C and carefully add it to the denatured intermediate chromosome specimen. Because the probe is both directly and indirectly labeled, it should be handled quickly and prolonged exposure to light conditions should be avoided. 2. Carefully cover the coverslip, taking care not to create air bubbles. If bubbles appear, gently press the coverslip to squeeze them out. 3. Seal the edges of the coverslip with rubber cement. 4. Place in hybridization cassette (see Note 13) and place in a plastic bag (optional) and hybridize for 48 h at 37°C. 1. Preheat all solutions in a 45°C water bath. 2. Remove the slides from the hybridization cassette and carefully remove the playdough. The coverslip may be removed with the slime or left on the slide. If the coverslip remains on the slice, place the specimen directly into the first vat of elution solution. 3. Wash 3 times in 50% formamide/2 X SSC for 5 min each time. the coverslip should slide off during the first elution. Gently shake the staining vat for a few seconds (see Note 14). 4. Make 3 washes in 1 X SSC for 5 min each and shake gently. 5. Immerse the specimen in 0.01% Tween-20/4 X SSC, remove the specimen and shake off the excess liquid but keep the surface of the slice moist. 6. Take 30~40uL of the sealing solution from Bottle 2 of the SKY kit (supplied by Applied Spectral Imaging) and add it to the specimen, cover with a coverslip. 7. Place the specimen back into the hybridization box and incubate at 37°C for 30 min. 8. Carefully remove the coverslip, take 30~40pLSKY test solution from bottle 3 of the kit (provided by Applied Spectral Imaging) and add it to the slice, and cover the coverslip. 9. Place the specimen back into the hybridization box and incubate at 37°C for about 30 minD 10. Carefully remove the coverslip and wash 3 times in 0.01% Tween-20/4XSSC for 5 min each time with gentle shaking. 11. Shake off excess liquid and add 30~40ul of test solution from bottle 4 of the SKY kit (supplied by Applied Spectral Imaging) to the specimen and cover with a coverslip. 12. Place the specimen back into the hybridization box and incubate at 37°C for 30 min. 13. Carefully remove the coverslip and wash the slide 3 times in 0.01% Tween-20/4XSSC for 5 min each time with gentle shaking. 14. Rinse the slides in 2XSSC at room temperature. 15. Seal the tablets with DAPI/anti-quencher in 15-20 bucket vials 5 and cover the coverslips to avoid air bubbles. 16. The slides are ready for reading. Specimens should be stored at 1 20°C. 1. Capture and analyze images as promptly as possible. Although fluorescein can be stored for several months at 20°C and in as little light as possible, the signal strength will diminish over time (see Notes 17-20). 2. Train to capture and analyze images with the system when it is installed in the laboratory. 1. Remove residual mirror oil from G-banded specimens by washing twice in xylene for 5 min each time at room temperature (see Note 15). 2. Immerse the specimen in methanol for 10~15 min to discolor, or until the specimen is completely discolored. 3. After discoloration, the specimen is subjected to a series of ethanol rehydration: 95%, 80%, and 70% ethanol, each time for 5 min, and left to dry. 4. Continue from step 5 of 3.3 of this chapter. 5. If the specimen has been previously banded, the denaturation time in 70% formamide/2 X SSC should be adjusted to reflect the specimen characteristics (see Note 16 and Table 1). A conservative denaturation time of 40 s is used. For more product details, please visit Aladdin Scientific website.
Tissue Culture Media Colchicine Citrate KCl Methanol Ice Ethanol Deionized Formamide 20 X SCC HCl PBS MgCl2 Dispensing Labeling Probes Xylene
Dissecting knives and blades Sterile Petri dishes Tissue culture flasks Incubators Water baths Centrifuge tubes Pipette guns Staining cylinders Fluorescence microscopes
