Chromosome Microdissection Experiment
Chromosome Microdissection Experiment
It has been well known in recent decades that chromosomal rearrangements are present in most human tumors and in certain specific human genetic diseases. Tumor-specific chromosomal abnormalities result in activation of proto-oncogene products, production of tumor-specific fusion proteins, or inactivation of tumor suppressor genes. Since the establishment of chromosome-banding techniques, cytogenetic analysis of nonrandom chromosomal abnormalities in tumors has become an indispensable indicator for the diagnosis and prognosis of many human tumors , but routine chromosome-banding analysis does not identify all cytogenetic chromosomal rearrangements (e.g., complex chromosomal rearrangements, minicircular chromosomes, and emergent nonequilibrium translocations that are not identifiable). Limitations of this technique have hampered the determination of karyotype in many human tumors, especially solid tumors, but this situation has been remedied by chromosome microdissection and FISH techniques. Chromosome microdissection has emerged as a powerful tool for rapid transition from cytogenetic analysis to molecular detection.
Operation method
microdissection of chromosomes
Materials and Instruments
Biotin-16-dUTP 13U ul Sequencing enzyme Affinity factor-FITC and anti-affinity factor Move I. Preparation of chromosomes in the middle of microdissection 1. Take 0.5 mL of peripheral blood and mix it with 4.5 mL of RPMI1640 culture medium containing PHA, and incubate the cells at 37°C for 64~68 hours. 2. Add 25 ml of colchicine (lO^g/mL) to the culture medium 20 min before harvesting the cells, and continue to incubate at 37℃. 3. Transfer the cell culture to a 15 mL centrifuge tube and centrifuge at 250 g for 5 min, then carefully discard the supernatant. 4. Resuspend the precipitate with 10 mL of hypotonic solution, incubate at 37°C for 12~18 min, centrifuge at 250 g for 5 min, and discard the supernatant. 5. Gently resuspend the cells with 8 mL of freshly prepared Camoy fixative, place the tube on ice for at least 2 h, centrifuge at 250 g for 5 min, and then carefully discard the supernatant. 6. Wash cells twice with 5 mL of fixative and centrifuge at 250 g for 5 min. 7. Resuspend cells with 0.5~1.5 mL of fixative to obtain a slightly opaque suspension for titration (see Note 2). 8. Place 2 or 3 drops of cell suspension on a clean coverslip tilted at a 45° angle and spread the entire slide with the flow of liquid; do not blow the slide to avoid contamination. 9. Place the slides in a sterile container at 37°C for 3 to 5 days (see Note 3). 10. Complete the standard G-strip treated with Trypsin-Giemsa before microdissection. Treat with Trypsin Working Solution for 1-2 min at room temperature/transfer the slice to Giemsa Working Solution at room temperature for 5 min, then rinse the slice with distilled water and air dry. 1. Locate the target chromosome under an inverted microscope that has a freely rotatable stage so that the target chromosome is perpendicular to the axis of movement of the glass needle. If possible, the microscope should be placed on a shock-proof stage to minimize vibration. 2. Cut the area of the target chromosome with a glass needle controlled by a micromanipulator fixed to the microscope (see Note 4). 3. Place the tip of the glass needle directly above the target DNA fragment, then move the glass needle downward to gently touch the fragment. Use electrostatic force to draw the DNA fragment to the tip of the needle, then move the tip of the needle into 5ul of collection solution. 4. After collecting the desired number (5 copies) of cut DNA fragments, place a drop of mineral oil over the collection solution. Prior to amplification of microdissected DNA, deconvolute the very tightly packed DNA by treating with topoisomerase I at 37°C for Ih. 1. Perform the PCR initiation program (5~8 cycles, 94°C, lmin, 30°C, 2 min, 37°C, 2 min), and add about 0.3U of sequencing enzyme at 30°C in each cycle (13 U/mL of sequencing enzyme diluted 1~8 times with enzyme dilution buffer, and 0.2PL of sequencing enzyme dilution was added to 5uL of the reaction system). 2. After completing the above pre-amplification steps, perform the conventional PCR reaction with Taq enzyme in the same PCR tube, the PCR system is 5uL, the PCR reaction conditions: 94°C, lmin, 56°C, lmin, 72°C, I.5 min for 30 cycles, and finally extended at 72°C for 5 min. 3. Add 2 uL of the first PCR product to a separate 50-ul PCR system and perform 20 PCR cycles under the same conditions as in step 2. 4. The success of the process can be recognized by electrophoresis, where 5ul of the PCR product is electrophoresed on a 1% agarose gel. The size of the PCR product should be in the range of 200-800bp. 1. The FISH probe was prepared by adding the second round of PCR products to the same 50-bucket PCR system as above (except that a final concentration of 20 umol/L biotin-16-dUTP was added) and the PCR reaction was continued for 20 cycles: 94°C, l min, 56°C, 1 min, 72°C, 2 min, and a final extension of 5 min at 72°C. The PCR reaction was then repeated for 20 cycles at 94°C for 2 min, and for 2 min at 72°C for 2 min. 2. Centrifuge the PCR products on a Bio-gelP6 filtration column as described in the product insert to remove undoped biotin-16-dUTP. 3. The probe was precipitated with 1/10 volume of 3 mol/L sodium acetate and 2-fold volume of ethanol for 20 min at 4°C. The probe was centrifuged at 10,000 g for 15 min at 4°C and resuspended in 40!uLTE buffer. 4. FISH probe hybridization is based on the previously described procedure [12]. Briefly, for each hybridization reaction, approximately IOOng of labeled probes were mixed in IOpL of hybridization buffer (7pL of hybridization mix, 2 buckets of probes, and lyL of human Cotl-DNA), denatured at 75°C for 5 min, followed by pre-hybridization at 37°C for 20 min. 5. Intermediate chromosome specimens are denatured in Denaturing Solution at 72°C for 2 min and then dehydrated in a series of 70%, 85% and 100% ethanol. 6. Place a drop of hybridization buffer containing the probe on the denatured slides, cover with 22 mmX22 mm coverslips and seal the slides with rubber adhesive. Transfer to a wet box at 37°C for overnight hybridization. 7. After hybridization, remove the coverslip and wash the slides three times with Wash I at 45°C for 5~1Omin each time. 8. Wash the slides 3 times with Wash II and once with Wash III at room temperature for 2 min each time. 9. Hybridizations containing only directly labeled probes can be analyzed at this point. For hybridizations with biotin-labeled probes, treat with 40 ML of PNM buffer containing 5 x g/mL FITC-affinity for 20 min at room temperature, then wash the specimen as in step 8. 10. Chromosome specimens are treated with 40 buckets of PNM buffer containing 5ug/mL of anti-affinity antibody for 20 min at room temperature to amplify the fluorescent signal; then the specimens are washed as in step 8. 11. Chromosome specimens are usually treated with another layer of FTTC-affinity, under the same conditions as in step 9. 12. Chromosome specimens are dehydrated in 70%, 85% and 100% ethanol series and dried in air. They are restained with 40juL of DAPI (ljug/mL), covered with a coverslip, and visualized with a fluorescence microscope fitted with appropriate filters. Caveat 1. In order to minimize the effects of contamination, all glassware containing reagents should be washed and autoclaved twice before use, and high-purity water should be filtered through a 0.22um filter and autoclaved twice for 40 min. High purity water should be filtered through a 0.22 um filter and then autoclaved twice for 40 min. Similarly, salt solutions should be prepared with high purity reagents, following the method of water filtration and autoclaving. 2. Fix cells with Camoy fixative (methanol:glacial acetic acid=3:1); cells preserved in fixative for 1 year will yield high quality FISH probes. 3. Intermediate chromosome specimens used for microdissection should preferably be 3?14d old; exposure of specimens to high temperatures (>60°C) or prolonged storage may reduce the quality of the dissected products. 4. Glass needles are prepared using an automated, spring-loaded pipette former. The diameter of the tip break corresponds to the width of the chromosome. The needles are stored in a sterile petri dish and irradiated with a UV lamp for 5 min before each use to ensure that DNA contamination is eliminated. 5. DNA contamination is a critical issue in the amplification of cleaved DNA. Since the amount of DNA initially cleaved is very small (1(T13?KT14 g/fragment), even a small amount of DNA contamination can overwhelm the cleaved DNA and result in a useless PCR amplification product. Contaminating DNA can come from the glass needle coming into contact with other DNA, from the air when the cap is repeatedly opened and closed, or even from contamination of exogenous DNA in the reagents prior to cutting. Therefore, all reagents used for microdissection should be tested for contamination before use. The purity of reagents for PCR can be checked by electrophoretic analysis after 40 cycles of plain PCR with o.Ing human whole genomic DNA or without DNA: 94°C, lmin, 56°C, lmin, 72°C, 2 min. When the positive control product is 200?800bp and the negative control product is little or no visible product, these reagents are suitable for microdissection; otherwise, each reagent in the system is replaced until a negative blank is obtained. After all PCR reaction reagents have been confirmed to be suitable for microdissection, topoisomerases and sequencing enzymes should also be tested. For more product details, please visit Aladdin Scientific website.
RPMI 1640 complete medium PHA colchicine amine hypotonic solution Fixation solution Trypsin working solution Gemma staining solution Sodium acetate Enzyme dilution buffer PCR reaction mixture PNM buffer
Glass Needles Kopf Needles or Pipette Shapers Stratalinker 2400 Inverted Microscope Hydraulic Micromanipulators PCR Instruments PCR Tubes