Multiplex telomere fluorescence in situ hybridization assay
Multiplex telomere fluorescence in situ hybridization assay
The multiple telomere FISH technique described in this chapter is the Chromoprobe Multiprobe-T system developed by Cytocell, which uses a microscope slide divided into 24 square zones and a Multiprobe mount that functions as a 24 square protruding block for coverslips. The system can handle 24 two-color (48) hybridization reactions simultaneously, so all telomere detection can be done on a single slide. Source: Molecular Cytogenetics Technology and Applications
Operation method
Multiplex telomere fluorescence in situ hybridization assay
Materials and Instruments
Immortalized Lymphocyte Cell Lines Move I. Preparation of fixed mid-stage chromosome cell suspension (1) Peripheral blood samples 1. Preheat RPMI-1640 medium, FCS, L-glutamine and penicillin/streptomycin in a 37℃ water bath. 2. In an ultra-clean bench for secondary cell culture, prepare complete culture μ-medium under aseptic conditions: 100mL FCS, 5mL L-glutamine, 5mL penicillin/streptomycin, and add RPMI-1640 to 500 ml. 3. In each of 2 Nunc culture tubes, add 0.25mL of peripheral blood, 5mL of complete culture medium, and PHA (final concentration of 2%). 4. Incubate at 37°C in an incubator for 72h. 5. Add 10OpL of 15mg/mL thymidine to each culture and mix gently. 6. Incubate at 37℃ in an incubator for 16~18h. 7. Centrifuge at 180g for 5min. 8. Discard the supernatant and resuspend the cells with 5mL of complete culture. 9. Centrifuge at 180g for 5min. 10. Discard the supernatant and resuspend the cell pellet with 5mL of complete culture. 11. Incubate at 37℃ for 5h. 12. Add 100/×L10jLtg/mL KaryoMa x colchicine. 13. Incubate at 37°C for 10min. 14. 180g-centrifuge for 5min. 15. Discard the supernatant and suspend the cell clumps with 7mL of 0.075 mol/L KCl pre-warmed at 37°C. 16. Incubate at 37°C for 15min. 17. Centrifuge at 180g for 5min. 18.Discard the supernatant. 19.Place the tube on a vortex shaker at medium speed and slowly dropwise add pre-cooled methanol:ice acetic acid (3:1) fixative to the cells. When the cell suspension turns brown, accelerate the dropwise addition of fixative to a total volume of 5mL. 20. Centrifuge at 180g for 5min and discard the supernatant. 21.Add 5mL of pre-cooled fixative. 22. 180g-centrifuge for 5min, discard supernatant. 23. Add 5mL of pre-cooled stationary solution. 24. Centrifuge at 180g for 5min, discard supernatant. 25. Add 3mL of pre-cooled fixative to resuspend cells. 26. Store at -20℃ for backup. (2) From immortalized lymphocyte cell lines 1. proliferate cells until whole medium to 50mL. . 2. 18~24h before harvesting cells, replace with fresh culture medium. 3. At harvest, transfer 20mL of well-grown cells to a 50mL centrifuge tube. 4. Add 200ΜL of colchicine at a concentration of 10μ×g/mL and mix gently. 5. Incubate at 37℃ for 50~60min. 6. Centrifuge at 180g for 5min. box or water bath). 5. Preheat the Multiprobe® device with the probe face up (i.e., protruding surface facing up, labeled surface facing down) and do not touch the surface of the protruding square block. 6. Prepare one staining vat with 2 x SSC and three staining vats with 70%, 85% and 100% ethanol series. 7. Wash the piece of spotted samples with 2×SSC for 2min, and then dehydrate it in 70%, 85% and 100% ethanol for 2min each in turn. 8. The slices were air dried and placed on a 37°C heating plate with the sample side facing up. 9. At the same time, take 2,preheated spots of hybridization solution with a 10 bucket pipette onto the protruding square block on the Multiprobe® device (placed on the 37°C heating block). 10. Carefully flip the piece (dot sample side down) to make contact with the corresponding square on the Multiprobe® device assembled to the Multiprobe® device; i.e., square 1 is in the uppermost right corner of the Multiprobe® device. 11. Carefully lift the assembled unit and turn over so that the sheet is underneath the Multiprobe® unit. 12. Place on a 37°C heating plate for 10min. 13. Carefully transfer the assembled device, keeping it horizontal, to a 75°C heating plate. 14. Denature for 1~3min, the denaturation time depends on the length of the immobilized chromosome suspension. 15.Immediately transfer the assembled device to a preheated ChromoprobeMultiprobe® hybridization chamber (or light-avoiding plastic slide cassette), replace the lid, and leave overnight in a de-capped water bath. ii, (4) Post-hybridization elution and sealing of slides 1. Pour the elution solution I into a clean staining vat and pre-warm it to 72°C in a water bath before adjusting the pH to 7.0. 2. Pour Elution Solution II into a clean staining vat and leave at room temperature (20~25℃). 3. Remove the assembled Multiprobe® device from the hybridization chamber. 4. Carefully remove the Multiprobe® device and immediately place the slice in the elution solution I for 2 min, use tweezers to Hold the slice and gently oscillate it up and down. 5. Transfer to Elution II for 30s. 6. Hold the long side of the slice and drain off the excess liquid on a paper for 10~20s, but take care that the surface of the slide does not dry out. 7. Add 20 μL of DAPI-containing anti-quencher solution to the ends of a coverslip (24 mm × 60 mm, supplied in the kit), and gently press the hybridized area on the slice onto the coverslip. 8. Examine the area of recombination and gently press with blunt-tipped tweezers to expel air bubbles. 9. Place absorbent paper over the top of the specimen and press gently to remove excess liquid. 10. Apply nail polish around the coverslip to seal the slide. 11. Leave the specimen in the black box or slide holder for at least 10 min before observation for reading. (5) Fluorescence microscopy to analyze the slides after hybridization 1. Place the specimen on the microscope carrier stage. 2. Observe the first square using a 10x objective and DAPI filter to observe the mid-stage chromosomes of individual cells. 3. Add mirror oil to the square block and convert to 100x oil lens. 4. Convert to a three-pass filter (DAPI/FITC/Texas Red) to observe mid-stage chromosomes. 5. count the green short arm signal and the long arm red telomere signal and record the position of the chromosome. The normal experimental result for the first square is that on chromosome 1 there are two green signals in the short arm telomeric region and two red signals in the long arm telomeric region. 6. Confirm the experimental results with FITC filter or Texas Red filter, respectively. 7. Repeat steps 2 to 6 until the results are clear for each of the five different mid-split phases in the well. 8. Repeat steps 2 to 7 for each well of the assay. 9. Record the results of the analysis. Any abnormal results or unsatisfactory hybridization results with certain probes should be redone with those probes. 10. Confirmation of the abnormality by using fresh samples from the patient and, if possible, analyzing parental samples will help to determine whether the abnormality is a true abnormality (and, if true, whether it is primary or familial) or the result of a benign polymorphic variant. Extraction of telomere cloned DNA 1. Prepare LB plates containing specific antibiotics (50μg/mL ampicillin, 35μg/mL kanamycin, or 12.5μg/mL chloramphenicol). 2. Remove the glycerol preservation tube from -70°C and place on dry ice. 3. Take the glycerol-preserved strains and streak them on the LB plate, then incubate them overnight in a 37℃ bacterial incubator. 4. Add 100mL of 2×YT medium to a 500mL centrifuge tube and add antibiotics at the concentration shown in step 1. 5. Select single clones from the plate to inoculate into 2×YT medium and incubate overnight at 37℃ in a shaker. 6. Take 700 μL of bacterial culture and 300 μL of 100% glycerol and mix it well, divide it into two Eppendorf tubes, label it well and store it at -70℃. 7. Centrifuge the remaining bacterial culture at 350g for 15min. 8. Discard the supernatant and drain the centrifuge tube upside down on paper. 9. Suspend the precipitate with 30mL of cold cell resuspension solution, mixing and aspirating vigorously several times so that the small cell clumps are thoroughly suspended. 10. Add 30mL of cell lysate and gently invert it up and down once. 11. Leave at room temperature for 5min (do not extend time). 12. Immediately add 30mL of cold neutralization solution, gently invert up and down to mix. 13. Leave on ice for 15min, and invert up and down several times every 5min. 14. Centrifuge at 4000g-centrifugation for 40min at 4°C. 15. Label a new 250mL centrifuge tube and place a plastic funnel on top. 16. Cut a round hybridization mesh sieve with a slit cut in the center and fit it onto the funnel. 17. Carefully pour the supernatant from the centrifugation onto the mesh sieve, and the liquid is collected through the mesh sieve and funnel into the 250mL centrifuge tube, taking care that no flocculent material passes through the mesh sieve. 18. Remove the funnel and mesh sieve and wash with water and ethanol for next use. 19. Add 0.7x volume of anhydrous isopropanol to precipitate DNA. 20. Mix well and centrifuge at 4000g for 10min. 21. Discard the supernatant, add 25mL of 70% ethanol. 22. Shake and mix well, centrifuge at 4000g for 10min. 23. Discard the supernatant and invert the centrifuge tube onto a piece of paper to remove excess liquid. 24. Resuspend the DNA precipitate with 1mL TE (pH 8.0). 25. 5 μL of extracted DNA was detected by electrophoresis on a 0.9% gel, using Xmnd III DNA as a as a reference standard, the size of the intact mucilage should be at the position of the large molecular mass. 26. Measure the concentration of extracted DNA (e.g., using a fluorescence photometer). 27. Store the extracted DNA at -20°C. IV. Indirect labeling of telomeric cloned DNA by notch panning method 1. Place the Eppendorf tube on ice, then add 1.0 μg of DNA to be labeled, 1.2 μL of biotin-16-dUTP (if labeled with short-arm probe) or digoxin-11-dUTP (if labeled with long-arm probe), etc., and add distilled water to a total volume of 50 μL. 2. Shake gently and centrifuge slightly to remove air bubbles. 3. React for 90 min in a 15°C water bath. 4. Place the reaction system on ice to pause the reaction. 5. Take 5 μL on a 2% agarose gel to detect the fragment size and use Phi×174 as a reference standard for DNA size. 6. The most suitable fragment size for hybridization is 50~500bp bands. If the fragment size is larger than 500bp, add 1.0μL DNase I and incubate at 15℃ for 30min. 7. When the probe size is appropriate, remove unbound nucleotides by centrifugation on a SELECT-B column containing cross-linked dextran G50. 8. Add 25 μL of salmon DNA (if the labeled probe is more than 1 wow, add salmon DNA proportionally. 9. Add distilled water to adjust the probe concentration to 10ng/μL (the amount of probe already used for detection should be deducted). 10. Record the volume of 10ng/μL probe mixture. 11. Add an appropriate amount of human Cot-1 DNA per microliter of probe mixture [the amount to be added depends on the size of the probe and the original inserted fragment. 12. Calculate the volume of human Cot-1 DNA/probe mixture containing 120 ng of probe and remove and transfer to a new Eppendorf tube by volume. 13. Dry the Cot-1 DNA/probe mixture by centrifugation in a vacuum centrifuge (be careful not to over-dry it). 14. To obtain a hybridization solution mixture of telomere-specific probes for the long and short arms of a chromosome, resuspend the Cot-1 DNA/long or short arm probe mixtures in 38 μL of hybridization solution, and combine the two to form 76 μL of hybridization solution. 15. If preparing probes to detect long or short arm telomeres alone, resuspend the Cot-1 DNA/probe mixtures directly in 76 uL of hybridization solution. 16. After mixing, store at -20°C. V. Hybridization of indirectly labeled probes and post-hybridization treatment (1) Hybridization 1. Turn on the heating plate, adjust the temperature to 75℃ and preheat. 2. Take 18 buckets of probe hybridization solution into Eppendorf tube and preheat to 37℃ (water bath or heating plate). 3. Adjust the cell suspension to the optimal concentration as shown in 3.2.1. 4. Soak high quality microscope slides in anhydrous methanol for 2 min. 5. Dry the slides with clean soft paper. 6. 10~20;×L of the adjusted concentration of cell suspension was added dropwise to the center of the slide. 7. Leave the specimen to dry and check the quality of the specimen with a 10x objective of a phase contrast microscope. 8. Mark the experiment on the abrasive and use a diamond pen to demarcate the sample area on the back of the slide. 9. Prepare one staining vat for 2 x SSC and three staining vats for 70%, 85% and 100% ethanol in that order. 10. The prepared slices (spotted with fixed cell suspension) were washed with 2×SSC for 2 min, and then dehydrated in 70%, 85% and 100% ethanol for 2 min each in sequence. 11. After air drying of the slice, with the sample side up on a 37°C heating plate 12. Gently aspirate up and down to mix the preheated hybridization solution, being careful not to create bubbles. 13. Aspirate the hybridization solution and add it to the center of the area delineated by the diamond pen. 14. Carefully place a 22mm x 22mm coverslip over the hybridization solution and gently press with blunt-tipped tweezers to dislodge air bubbles. 15. Carefully place the slice (coverslip side up) on a 75°C heating plate. 16. Denaturation, 1~3min, denaturation time depends on the aging of the cell suspension 17. Immediately after denaturation, transfer to a preheated light-proof plastic slide box and hybridize overnight in a 37°C water bath (water bath uncovered). (2) Post-hybridization elution, antibody detection and re-staining 1. Prepare containment solution and antibody solution 1~3. 2. Shake the antibody solution and centrifuge it in a microcentrifuge at maximum speed for 10 min. keep it away from light and pre-warm it at room temperature if necessary. 3. Add Eluent I to a clean staining vat, pre-warm to 72°C in a water bath and adjust pH to 7.0. 4. 4. Add Elution Solution II to a clean staining vat and leave at room temperature (20~25℃). 5. Meanwhile, place absorbent paper moistened with water on the bottom of an empty light-proof plastic slide box (e.g. empty slide box). 6. Using scissors, prepare four sheets of sealing film for each specimen measuring approximately 22 mm × 64 mm. 7. remove the specimen from the light-proof plastic box and quickly remove the coverslip on a piece of paper. 8. Immediately place the slides in elution solution I for 2 min, hold the slides with forceps and gently oscillate them up and down. 9. transfer to eluent n for 30s. 10. Hold the long side of the slice and drain off the excess liquid on a paper for 10~20s, taking care not to over-dry the surface of the slice. 11. Rack the slice into a pre-prepared light-proof plastic slide box (e.g., the ends of the slides are resting on glass rods or on the platform at the bottom of the box), take 300 μL of the sealing solution and add it to the slice, and cover it with a sealing film to avoid air bubbles. 12. Cover the slide box with a lid and incubate at 37℃ for 40~50min. 13.Preheat 1.5L of ST buffer at 37℃. - 14. Remove the sealing film after the incubation is completed and place the slides on a specimen holder to soak in the preheated ST buffer. 15. Place on the shaker platform and oscillate for 3 min, and place the inverted lightproof container on top to maintain a dark environment. 16. Drain excess liquid from the specimen as shown in step 10. 17. Place the slice on a shelf in a lightproof plastic slide box, and take 80 μL of antibody solution 1 and add it to the hybridization area. 18. Cover with sealing film to avoid air bubbles 19. Cover with a lid and incubate for 10min at 37℃ in a warm box. 20. Remove the sealing film and place the slice on a specimen holder immersed in pre-warmed ST buffer. 21. Place on the shaker platform and oscillate for 3min under light protection. 22. Pour off the original ST buffer and replace with new preheated ST buffer. 23. Repeat steps 21 and 22 2 times. 24. After 3 washes, drain off the excess liquid as shown in step 10. 25. Place the slice on a rack in a plastic light-proof container and take 5O μL of antibody solution 2 and add it to the hybridization area. 26. Cover with sealing film to avoid air bubbles. 27. Cover with a lid and incubate for 10min in a 37℃ temperature chamber. 28. Remove the sealing film and wash 3 times in hot new ST buffer for 3min each time. 29. Drain the slices of excess liquid as shown in step 10. 30. Place the slice on a rack in a plastic light-proof container and take 80 μL of antibody solution 3 and add it to the hybridization area. 31. Cover the lid and incubate for 10 min at 37°C in a warm box. 32. Remove the sealing film and wash 3 times in hot fresh ST buffer for 3min each time. 33. During the final ST buffer elution, add about 25μLDAPI re-staining solution to a 22mm×22mm coverslip placed on paper. 34.Drain the slice of excess liquid as shown in step 10. 35. Gently invert the hybridized area of the slice onto a coverslip spiked with DAPI re-staining solution. 36.Place an absorbent paper over the specimen and press gently to remove the excess of the re-staining solution. 37. Examine the restained area and remove air bubbles by pressing gently with blunt-tipped forceps. 38. Apply nail polish around the coverslip to seal the slide. 39. Place the slides in the light as shown in II. (5) until dry and observe with a fluorescence microscope. Caveat 1. To prepare high-quality chromosome specimens, it is best to collect peripheral blood samples using lithium heparin anticoagulant tubes; samples should be transported at room temperature and cultured in exhaustive blocks. The quality of fixed cell suspensions decreases significantly with storage time. 2. Closure solutions can be prepared in large quantities, filtered and dispensed, and stored at 4°C for up to 6 months. 3. 1mL of antibody solution can be stored at 4℃ away from light for 1 month. 4. To avoid contamination, cell culture medium should be used in a water bath specialized for cell culture. 5. Colchicine prevents spindle formation and blocks cell chromosomes at mid-cell division. The time of cell treatment with colchicine affects the morphology of chromosomes. If the treatment time is too short, the chromosomes may be longer, resulting in a lower division index; if the treatment time is too long, the chromosomes will be too short for analysis. 6. When the hypotonic treatment time is not long enough, the chromosomes cannot be released from the cells. Too long a treatment time will cause a large number of cells to rupture prematurely. 7. Although the probes and reagents supplied with the ChromoprobeMultiprobe®-T kit have been quality-checked by Cytocell, it is recommended to optimize the kit hybridization conditions by pre-testing with a high-quality normal control chromosome suspension before hybridizing the very important samples to be tested. 8. Although ideally the splitting index should be >5%, there are sometimes problems that prevent this from being achieved, e.g., the length of sample preparation or the source (e.g., mitosis in leukemia samples is often insufficient). In such cases, it should be ensured that there are approximately 5 mid-phase divisions in the central region of the specimen. When some samples have very low division indices and there are few samples, you should choose to analyze the samples using a multicolor FISH technique such as MTEL. 9. If there are not enough midphase chromosomes on just a few square blocks, the sample can be re-spotted at the same location where the original sample was taken. 10. It is possible to flip the specimen heating block so that the top is flat as a 37°C heating plate. However, the 75 ℃ electric thermostat plate must be solid and the surface temperature is uniform. 11. It is best to hold the slice/Multiprobe device in the frosted position with the index finger and thumb of one hand to lift the slice/Multiprobe device, then hold (do not squeeze the device) the slice/Multiprobe device with the index finger and thumb of the other hand and flip it over, either with the palm of the hand or with a pallet rest to keep it horizontal for transfer. 12. Correct denaturation is important. If the chromosomes are denatured for too long, the chromosomes will swell and potentially leave a probe signal along the chromosome arms (Fig. 2C). Conversely, denaturation for an insufficient period of time maintains the chromosome morphology but is inefficient for hybridization if it can be hybridized on. In summary, the longer the cell suspension is stored at -20°C, the longer the chromosome denaturation time should be. For example, specimen preparation and hybridization on the same day, then the denaturation time is only 45s, however, a sample left for more than a year, the denaturation time is greater than 3min. preparation time for 1 ~ 3 months of samples at 75 ℃ the average denaturation time is 1min, 15s. 12.- The stage of some microscopes makes it difficult to observe the end of the slice. If this is the case, the end can be easily observed by rotating the slice by 180°.14. Before recording the results, you should familiarize yourself with the characteristics of the telomere clones used to avoid misinterpretation. It is normal for certain clones to be used for polymorphism detection that hybridize only to the corresponding homologous telomeres, but if polymorphism is present, these clones hybridize only partially or not at all to the homologous chromosome. In addition, some clones are characterized by cross-hybridization, and these clones hybridize to other telomeres in addition to the homologous telomeres. By way of example, a clinically significant unbalanced telomere rearrangement is shown in Fig. 2, which shows a normal 3p3q hybridization pattern, a 2q probe polymorphism, and a 12p cross-hybridization. Table 2 lists the known polymorphisms and cross-hybridization patterns of first- and second-generation telomeric clones.15. When experiments must be repeated, it should be determined whether it makes sense to use the entire new Multiprobe® slide and apparatus. Other options include: (i) re-purchasing individual telomeric probes that have been labeled (Cytocell or AppligeneOncor or Vysis); (ii) obtaining a set of glycerol-preserved telomeric clones and preparing the DNA and probes for hybridization yourself. The latter approach is more economical, and once the clones are amplified the probes can be labeled and stored in bulk; suitable probes can be stored at -20°C for at least 2 years. Table 1 lists which first- and second-generation telomeric clones are available and where glycerol-preserved strains or puncture cultures of these clones can be obtained; it also labels the telomeric clones used with the ChromoprobeMultiprobe®-T kit.Some unusual results may reflect true subtelomeric rearrangements or telomere polymorphisms, but it is important to avoid misinterpretation at this point. Table 2 lists the polymorphisms and cross-hybridization patterns associated with the use of first- and second-generation telomere-specific cloning probes. Figure 2 shows an example of a FISH experiment17. it is not necessary to prepare telomere clone DNA if the Cytocell ChromoprobeMultiprobe®-T kit or other commercially available kits (e.g., Vysis TotelVys10nMulticolorFISHProbePanel kit) are used; however, it is possible to obtain and store a set of telomeric clones that can be used for a small number of replicate experiments. that can be obtained and preserved in case a small number of replicate experiments are needed. Table 1 lists what first- and second-generation telomeric clones are available and where glycerol-preserved strains or puncture cultures of these clones can be obtained, and the list in Table 1 also includes the telomeric clones used in the ChromoprobeMultiprobe®-T kit.18. If dry ice is not available, glycerol-preserved strains can be placed on ice for a shorter period of time, but repeated manipulation will reduce the activity of the glycerol-preserved strains.19. If the precipitate does not suspend quickly, it can be left at 4°C overnight.20. If using Cytocell ChromoprobeMultiprobe®-T series kits or other commercially available kits, this part of the procedure is not necessary.21. Because indirectly labeled probes can be stored for about 2 years, the amount of labeled DNA and the volume of the labeling reaction can be increased. The22. The actual concentration of DNAase may also vary from batch to batch, and the amount of DNase used varies from template to template. It is best to optimize the amount of enzyme and reaction time used for templates when using each new batch of DNAase, in case the same conditions are applied to label the same template probe in the future.23. Each batch of probe preparation should be optimized for the amount of Cot-1 used. the amount of Cot-1 used should be based on the amount of Alu sequence in the probe. For telomere clones, the amount of Cot-1 is generally 625 ng/juL of probe mixture for labeled sticky grains with an insertion fragment of 30-40 kb; for PAC and small BAC probes with an insertion fragment of less than 100 kb or so, the amount of Cot-1 is generally 1 wow/bucket of probe mixture; and for large BAC probes, the amount of Cot-1 can be added to 3 μL of probe mixture. Note that the exception is probe clones for chromosome 19, such as F20643 and 20283 which require the addition of 10 μL of probe mixture.24.If an electric thermostat plate is not available, chromosomes can be denatured in 10% formamide/0.1 mol/L EDTA/2×SSC at 70°C for 5 min followed by 3 washes with 2×SSC at 4°C and then dehydrated in a series of ethanol. Probe mixtures were individually denatured before addition to the specimen, 95°C for 5 min.25. Antibody solutions should be prepared correctly according to the needs of the monochrome or two-color assay, and the antibody concentration is very important. If the antibody concentration is too low, there may be no hybridization signal or the hybridization efficiency is very low. On the contrary, if the concentration is too high, a speckle-like background may appear and it is impossible to distinguish whether it is a homologous signal or background (Figure 2D). Since there may be differences in antibodies from different batches, it is best to optimize the concentration of antibody dilution by hybridizing a reliable control probe to a high-quality chromosome suspension before testing the official sample For more product details, please visit Aladdin Scientific website.
Penicillin Glutamine Fetal bovine serum Phytohemagglutinin Thymidine Colchicine KCl Methanol Glacial acetic acid Sodium citrate SSC Glycerol Biotinyl-16-dUTP Digoxin-11- dUTP 10 XdNTP Mixture
Ultra-clean bench Cell culture flasks Nunc tubes Culture medium Phase contrast microscope Staining cylinders Heating plates CCD microscope fitted with Pinkel filter wheel Chromoprobe Multiprobe®-T kit Constant-temperature shaker Aminobenzylpenicillin Kanamycin Chloramphenicol LB agar plates 2 X YT Culture medium