Experimental application of fluorescence in situ hybridization in the preimplantation diagnosis of aneuploidy
Pre-implantation Genetic Diagnosis (PGD), as a major new method of preventing genetic disorders, avoids the need for prenatal diagnosis and the possibility of termination of pregnancy. For people at high risk of genetic abnormalities, preimplantation genetic diagnosis selects normal embryos for implantation by testing the genetic makeup of the egg cell or embryo, unlike prenatal diagnosis, which is a direct test of the genetic makeup of the fetus and carries the risk of having to terminate the pregnancy with an abnormal test result.
Operation method
Fluorescence in situ hybridization in the preimplantation diagnosis of aneuploidy
Materials and Instruments
Ice acetic acid Methanol Bovine serum albumin Sodium citrate Hypotonic fluid Pepsin HCl Formalin SSC Formaldehyde buffer Move I. Preparation of PB1, PB2 and oocytes (after separation from oocytes and embryos) for FISH analysis Preparation of PBl and PB2 specimens 1. Prepare fresh fixative (methanol: glacial acetic acid = 3:1) in 50 mL culture flasks and keep it in the freezer for spare use. 2. Pull the tip of the capillary pipette over the flame of a small blowtorch with an inner diameter of about 50 pm; too large an inner diameter may result in the loss of the polar body. 3. Add a few drops of fixative and re-treat the pre-treated slide to remove any grease or dust that may be present, blot dry with dust free paper. 4. Use a Pasteur pipette to pipette a small amount of HPLC purified water onto the slide where the droplet can be suspended. 5. After successful removal of polaroids from oocytes or fertilized eggs, the polaroids can be viewed with a phase contrast microscope (10X, 20X objective) in a 20uL droplet of culture solution (droplet covered with mineral oil) in a petri dish on a micromanipulator [6]. 6. Once the polar body is positioned, a small amount of water is drawn through a stretched capillary pipette to make sure that the fluid flows in and out of the pipette. 7. After aspirating a small amount of water, gently draw the polar body into the capillary pipette moving it towards the watery hangable droplet slide and release the polar body by gently blowing it out of the capillary pipette until the polar body is seen in the circle in the center of the slide. Do not allow the polaroid to become fixed and affixed to the glass surface of the hangable droplet slide. 8. After rinsing in water for a few moments, reintroduce the polaroid with a capillary pipette. The oil around the polaroid can usually be washed away with water. Move the polaroid to a clean slide and gently blow out the polaroid-containing water droplet to release the polaroid. 9. When the water has partially evaporated, the polaroid begins to swell and flatten against the surface of the slide. Use the same capillary pipette to place a drop of fixative on the polaroid before the water has completely evaporated to disperse the chromatin. 10. Before the fixative is completely dry, add another drop of fixative and repeat as many times as necessary until the cytoplasm is completely dissolved, observing this process carefully and removing as much of the cytoplasm as possible (see Notes 1 and 5). 11. Using a diamond pen, draw a circle around the chromatin to locate it, taking care not to be too heavy so as to avoid the influence of glass shavings, and then forcefully draw another circle around the circle on the back of the slide, so that the circle can be easily found and the position of the chromatin located for analysis. 12. Label the slide with the patient's name, oocyte number, and appropriate information such as the number of chromatin blocks. 13. Add a few more drops of fixative to the slide and blow dry with an earwash ball. 14. Slides may be hybridized after being left at room temperature overnight or immediately; if hybridizing immediately, pre-treatment is recommended. Good results can be obtained within a more reasonable time frame for transferring embryos. 15. Use a colorfast marker to draw on the back of the slide where the polar chromosomes are located in order to determine where to add the probe. 1. Prepare fresh fixative (methanol: ice acetic acid = 3:1) in 50 mL culture flasks and keep in the freezer for later use. 2. Carve a circle on the bottom of a 35 mmX10 mm covered Petri dish to make it easier to find the oocytes in this area, and add 3 mL of hypotonic solution to the dish. 3. Pull a 25~50uL capillary pipette over the flame of a small blowtorch to make a pointed capillary pipette with an inner diameter of about 70um. 4. Inhale a small amount of hypotonic solution into the capillary pipette. 5. Under a dissecting microscope, locate the ovalbumin inside the microdroplet of culture solution and reconfirm that the inner diameter of the capillary pipette made is larger than that of the ovalbumin and can be used to aspirate the ovalbumin; then use it to gently aspirate the ovalbumin and transfer the ovalbumin to the hypotonic solution dish. 6. After 3-5 min of hypotonicity, transfer the ovalbumin into the tube onto a slide with a little hypotonic solution. 7. Observe the ovalbumin continuously under an inverted microscope until it is almost completely dry. Place a drop of fixative on the ovalbumin before the hypotonic solution is completely dry and crystals form. Continue to observe the cells and place a drop of new fixative before they are completely dry. This was repeated several times until the cytoplasm cleaved, leaving the nucleus behind. Whether cytoplasmic removal is complete directly affects hybridization (see Note 1). 8. Mark the position of the ovoid spheres with a diamond pen for easy localization after hybridization. 9. Indicate the appropriate information on the frosted end of the slide, add a few drops of fixative, and blow dry with a wash ball. 10. The slide can be prepped. Before adding the probe, mark the exact position of the nucleus of the oval sphere on the back of the slide with a colorfast marker to identify the area to which the probe will be added. 1. Wash in 2 X SSC (pH 7.0~7.5) at 37℃ for 10 min. 2. Fixed in 1% formaldehyde for 5 min at room temperature. 3. Wash in 1xPBS (pH 7.0~7.5) for 5 min at room temperature. 4. Treat in pepsin solution dissolved in 0.01mol/LHCl at 37℃ for 5 min. 5. Wash in 1xPBS (pH 7.0~7.5) for 5 min at room temperature. 6. Remove the slide, shake off the liquid and wipe off the residual PBS on the back side. 7. Dehydrate the slides in a series of ethanol (70%, 85% and 100%) for 1 min each at room temperature and air dry for hybridization. 8. For specimens requiring repeat hybridization, remove the coverslips and place the slides in methanol solution for 5 min before pretreatment to ensure that the chromatin is immobilized on the slides. 1. Three-color probe mixing: centrifuge the three centrifuge tubes containing probes, and then use a vortex oscillator to oscillate the ideal probe and LSI probe hybridization buffer for 10 s. Prepare 10juL of probe hybridization solution, that is, lfxL of each chromosome 13, 18, and 21 probes, and add it to the microcentrifuge tubes containing 7;xLLSI probe hybridization buffer, oscillate the mixing hook, and centrifuge the tubes. 2. For probes with only one chromosome, the probe mix consists of IyL probe, 2 pL of HPLC-grade water, and 7 pL of Hybridization Buffer. For CEPX and CEPY probe mixtures, the probe and CEP Hybridization Buffer are shaken and centrifuged.10/xLCEPX and CEPY Probe Mixture Workup consists of: I1^LCEPX. and CEPY probe mixtures, 2 tubes of HPLC grade water and CEP Hybridization Buffer. 3. MdtiVysion's 5-color probe mixtures are ready to use. Centrifuge and shake well before use. All working solutions of the probe mixtures can be stored at -20°C for several months (within the expiration date provided by the manufacturer). 1. Before hybridization, use a diamond pen to cut a 22 mmx30 mm coverslip into 8 mmx8 mm pieces and place them in a covered glass petri dish. 2. Cut the sealing film into 22 mmx22 mm pieces and place them inside the dish. 3. Prepare the hybridization wet box: soak paper towel with sterilized water and put it into a glass dish, then put a slide holder, cover it with a lid and put it in a 37℃ incubator for pre-warming. 4. Take out the working solution of the probe mixture to be hybridized from -20℃, centrifuge it for 10s and then shake it to mix well. 5. Use a micropipette to add 2uL of the working solution of the probe mixture to the labeled chromatin area on the slide, and then quickly cover the 8 mmx8 mm coverslip with tweezers, taking care that no air bubbles will affect the hybridization. If air bubbles appear, press the coverslip gently with tweezers to expel the air bubbles. 6. Seal the coverslip with rubber cement or sealing film. When using sealing film, be careful to press down around the coverslip to ensure that the sealing film adheres to the slide (see Note 2). 7. Place the slide on a slide hot plate at 69°C for 8 min to denature both the probe and the specimen DNA. The slide specimen is then quickly transferred to a preheated wet box at 37°C for hybridization (see Notes 3 and 4). The following two elution steps are commonly used in our laboratory and are formamide-free and time-saving. This elution procedure is suitable for 1 to 3 color probe mixtures and MdtiVysionPGT (chromosomes 13, 18, 21, X and Y). 1. Preheat the staining vat with 0.4xSSC/0.3% NP-40 (pH 7.4) solution in a water bath at 73°C. Check the temperature of the vat with a calibrated thermometer before eluting the specimens. 4. Transfer the specimen to a vat with 2XSSC/0.1% NP-40 (pH 7.4) and elute for 1 min at room temperature. This elution procedure is for use with the MultiVysionPB (Polar Body) Probe Assortment, which includes probes for chromosomes 13, 16, 18, 21, and 22. 1. Preheat the staining vat with 0.7XSSC/0.3% NP-40 (pH 7.4) solution in a water bath at 73°C. Check the temperature of the vat with a calibrated thermometer before eluting the specimens. 2. Prepare a vat of 2XSSC/0.1% NP-40 (pH 7.4) solution and set aside at room temperature. 3. Place 0.7XSSC/0.3% NP-40 (pH 7.4) in the vat immediately after removing the coverslips, and elute for 7 min after all specimens (up to 4 slides in the same vat) have been placed in the vat. 4. Transfer the specimen to a vat of 2XSSC/0.1% NP-40 (pH 7.4) solution and elute for 1 min at room temperature. 1. Add 10 ju of DAPI/anti-quenching mixture per slide to the hybridization zone and cover with a 22 mmX30 mm coverslip. 2. Use a paper towel or blotting paper to distribute the solution evenly and remove excess liquid from the slide (for MultiVysion 5-color probe mixtures, a special DAPI-free restaining solution is provided). 1. Hybridization signals are observed under the oil lens of a 600x fluorescence microscope. Since fluorescent signals are easily quenched by light, to minimize sudden light attenuation, all liquid manipulations containing incendiary light pigments, including the processing steps of specimens after hybridization, should be performed in low light. All steps that do not require light, including hybridization incubation and elution, should be performed under low light (see Note 2). 2. The fluorescence microscope is excited by a 100 W mercury lamp, and although the manufacturer recommends a lamp life of at least 200 h, we have found that hybridization signals under the microscope are weak after 175 h, resulting in misdiagnosis. Spare mercury lamp bulbs should be available. 3. In the multicolor probe set of MuItiVysionPGT, the signal is blue for chromosome X, gold for Y, red for chromosome 13, light green for 18, and green for 21 (see Notes 7 and 8). 4. In the multicolor probe set for MdtiVysionPB poles, chromosome 13 has a red signal, 16 is light green, 18 is violet-blue, 21 is green, and 22 is gold (Fig. 1) (see Notes 7 and 8). 5. The signals (red, green, blue, gold, light green, and DAPI) are observed through single-channel filters corresponding to the fluorescein labeled by the probes, while dual- or triple-channel filters are only suitable for identifying non-specific fluorescent signals or for counting signal penetration common to the signals of filament probes, which hybridize to the a-satellite repeats and have large, bright signals (see Note 7). 6. When determining the number of signals, attention should be paid to the size and brightness of each signal, especially when two signals are in close proximity. Chromosomes 13, 21, and 22 are identified with site-specific probes containing homologous sequences of the chromosome to be studied and unlabeled closed DNA suppressor co-sequences. The sequence-specific probe signals are dots (see Notes 6 and 7) compared to the probe that counts the chromosome 16, 18, X, and Y chromosome mitotic grains (which hybridize to a large number of Satellite Repeat Sequences). 7. For different chromosome signals that are close together or even overlap with each other, it is recommended to look carefully with both single- and dual-channel filters to identify them from non-specific hybridization signals. On the image, overlapping parts of signals of different colors may appear yellow or white due to color combinations (see Notes 7 and 8). When combining probes with three or more colors, an image analysis system is highly recommended. 8. Each chromosome in PBl contains two chromatids, and each chromosome should have two signals of the same size, which should generally be separated by a distance greater than the diameter of one signal (A, C, and E in Figure 1). However, if the two chromosomes are close together, the signals may be linked together as a fluorescent band (the signal for chromosome 16 in PBl shown in Figure IE), representing two chromosomes (see Notes 6 and 9). 9. Each chromosome in PB2 contains a single chromosome and therefore each chromosome detected presents a single fluorescent signal (A, C, and E in Figure 1). 10. Normally, the chromosomal signals to be studied in each oval nucleus are two single signals (B, D, and F in Fig. 1). However, the chromosome signals can be shown as double-dot signals due to different cell cycle stages, chromosome condensation and spreading. After replication, the signal of each chromosome may appear as pairs of signal dots less than one signal diameter apart, or as two small bands that overlap each other, both of which should be recorded as a single signal according to analytical criteria (see Notes 7 and 8). 11. If two signals are equal in size and brightness and the distance between them is greater than the diameter of the two signals, they are counted as two separate signals. However, the distance between two separate signals in a chromatin hyperintense nucleus may also be less than the diameter of the two signals, and this must be taken into account to avoid misdiagnosis. The size and brightness of the signal of each nucleus should be compared with other nuclei on the same slide to differentiate between true and nonspecific signals. Hybridization sites with weak fluorescence intensity, small or flat surfaces with no incandescent appearance are excluded from the count (see Notes 6 and 7). 1. Before hybridization, the chromatin of the ovalbumin, PB and PB2 must be completely exposed, free of residual cytoplasm and securely attached to the slide. Therefore, prior to adding the probe, polar body or ovoid specimens should also be pretreated to maintain morphology and remove residual cytoplasm. Cytoplasmic residue is the biggest factor affecting hybridization through the nucleic acid replication process and can lead to non-specific fluorescent signals affecting the judgment of results, which is an important issue to overcome. Inadequate chromatin exposure and cytoplasmic residues can show non-specific fluorescent signals after hybridization, making signal determination difficult or even impossible to analyze. 2. Standardization and quality control monitoring of all operational steps involved in FISH, including fixation, are critical to the quality and interpretation of hybridization signals. To ensure specificity, a control lymphocyte specimen should be hybridized with each batch of specimens. The 3. Depending on the type of hybridization probe used, hybridization times can range from 2 h (for mitotic probes or sequence-specific probes) to 16 h (for whole chromosome coating probes or subtelomeric probes). With the MdtiVysicm probe combination, without DAPI re-staining, the hybridization time is within 3 h. If the hybridization time is too long, the probes can be used for a longer period of time than the MdtiVysicm probe. If the hybridization time is too long, placenta DNA labeled with aqua in the probe mixture will give a strong background to the chromatin. It is recommended that the hybridization time should not exceed 2 h for MultiVysion probes for ovoid and 3 h for MultiVysion probes for polar bodies. 4. The Hybrite hybridization device from Vysis can be used to denature and hybridize nucleic acids simultaneously (up to 12 slides at a time) in place of a slide warmer and incubator. It is programmable from denaturation (69°C, 8 min) to hybridization (37°C, 3-16 h). A few microliters of water are added to each slot along the slide hotplate to ensure a humid environment, especially important for overnight hybridization. After adding probes to the slides, covering the coverslips and sealing the slides with sealing film they are ready to be placed in the Hybrite hybridization device. Close the lid, start the program, and do not move the specimen until the hybridization process is complete. Use a slide thermometer to monitor, for example, the accuracy of the melting temperature before starting the program to determine proper functioning. 5. If the PBr fixation is poor and there is cytoplasmic residue, the probe nucleic acids may not be able to reach the nucleic acids of the fixed specimen resulting in hybridization failure. If a new hybridization is formed, the signal may be weak and/or scattered into multiple spots, which is most often seen in excessively degraded first polar bodies. Inadequate chromatin fixation coupled with cellular residues may result in non-specific fluorescence after hybridization, making signal determination difficult or even impossible to analyze. 6. Although all conditions are controlled according to the standard, sometimes the signal of one or more probes will be more diffuse or even scattered into multiple points, which makes the analysis of the results more complicated. This is usually due to chromatin degradation. When the signals are diffuse or separated, it is important to consider the quality of the signals from both the PBl and PB2 assays. 7. Nonspecific hybridization is visible under two or more filters, in the same position, but is usually weak. When the fluorescence signal of the probe is particularly strong, the penetrating signal can be observed under other filters, but the signal is usually weak and non-stereoscopic. This phenomenon generally occurs with the CX Satellite Repeat Sequence Probes, which have a strong and diffuse signal. 8. Strong nonspecific hybridization can be excluded with appropriate single- or dual-channel filters. If the perinuclear cytoplasm of the ovoid is cleanly removed and there is no hybridization signal, and other nuclei on the same slide have signals, ovoid abnormalities should be considered. This is not uncommon, especially if there are significant differences in nuclear size or if the nuclei are fragmented. 9. In evaluating PBl fluorescence signals, taking into account post-fertilization emigration (e.g., 24 h after retrieval), the signals representing each chromosome may be further separated, as chromosomes segregate over extended periods of time. These pre-separated signals are considered normal and the number of signals is easier to determine (see gold signal for chromosome 22 in PBl in Fig. IA and chromosome 18 in PBl in Fig. IE). 10. The diagnosis of preimplantation aneuploidy in ovalbumin, PB1, and PB2 by FISH has been performed with high accuracy and reliability. This has been verified by confirmatory studies of whole embryos with expected abnormalities and by follow-up cytogenetic testing of more than 500 clinical pregnancies from approximately 2000 PGD cycles. For more product details, please visit Aladdin Scientific website.
Microscope slides Culture flasks Pasteur pipettes Micropipette holders Diamond pens Earwash balls Anatomic microscopes Inverted microscopes Miniature blowtorches Capillary pipettes Thermostats Microcentrifuges Vortex oscillators Centrifuge tubes
2. Prepare a vat of 2xSSC/0.l% NP-40 (pH 7.4) solution and set aside at room temperature.
3. Immediately after removing the coverslips, place them in 0.4xSSC/0.3%NP-40 (pH 7.4) and elute for 5 min after all specimens (up to 4 slides in the same vat) have been placed in the vat.
5. Remove the specimen from the elution solution and soak it in HPLC-grade purified water, then stand the specimen vertically in a dark place (e.g., in a drawer) on a paper towel to dry.
5. Remove the specimen from the elution solution and soak it in HPLC-grade purified water. Stand the specimen vertically in a dark place (e.g., in a drawer). Allow to dry on a paper towel.