It should be noted that we describe only micromanipulator-based microdissection. Laser capture microdissection, a powerful technique, is more often used in tissue isolation, and is less frequently used to isolate such small-mass items as chromosomes.
1. Inverted microscope (Zeiss Axiovert); an upright microscope is unsuitable because the glass needle will not fit between the objective and the cover slip. It should have phase contrast, and minimum two objectives: x10 and x100. It is unnecessary to have any image analysis capability. However, a free rotating stage is quite useful because it allows the chromosome to be oriented in a particular way.
2. Needles (Frederick Haer & Co.). These can be made or purchased. For simple, frequent chromosome microdissection, making them by pulling capillary tubes
(1.0 mm OD x 0.75 mm ID or 1.0 mm OD x 0.5 mm ID [both 100 mm in length] work well for dissecting chromosomes, and eukaryotic and prokaryotic cells) is the least expensive option. In the event more complex operations (such as microinjection) need to be undertaken, or if microdissection will be an infrequent operation, they can be purchased (e.g., from Eppendorf).
3. Needle puller (Sutter Instruments model P-30). It is unnecessary to use one with the full capabilities of making patch clamp needles; the simplest model extant is sufficient.
4. Micromanipulator (Eppendorf model 5171). There are several possibilities depending on the level of precision desired. Narashige and Eppendorf make excellent micromanipulators, over a wide array of cost. All are sufficient for the task, but spending more is helpful in that more capabilities, such as automatically returning to a metaphase spread after replacing a needle, are offered.
3.1. Slide Preparation
1. Soak cover slips in 100% methanol for 5 min to remove any coating or grime.
2. Remove and dry with a lint-free towel.
3. Store in a clean, dry place until needed.
3.2. Bacterial Cultures
1. Grow desired bacterial cell line in the proper liquid medium overnight at 37°C, approx 230 rpm.
2. Place a drop (approx 2 |L) of liquid culture onto opposing ends of a clean slide. Add a drop of 3:1 methanol:glacial acetic acid to each of these areas. Heat at 37°C until dry. Set slides aside until needed.
3.3. Cell Cultures
1. Grow desired cell line in the appropriate growth medium at 37°C, 5% CO2.
2. For metaphase cells: add 0.1 |g/mL of culture colcemid to the flask 4 h before harvesting (see Note 4). After incubation lift cells from flask and place in 15-mL Falcon tube.
3. Centrifuge the liquid culture for 5 min at 150^ in a benchtop centrifuge. Repeat this step until the supernatant is clear. Carefully remove it and discard. Add 8 mL of 0.075 KCl hypotonic solution to each falcon tube and gently resuspend using a plastic pipet. Recap the Falcon tubes. Set a timer for 20 min for Chinese hamster ovary cells, 30 min for rat and human lymphocytes/lymphoblastoid cells, or 60 min for mouse lymphocytes. Incubate cells in solution at 37°C.
4. When hypotonic is complete, add 1 mL of fixative. Mix gently and centrifuge for 5 min at 150^.
5. Remove supernatant, add 3 mL of fixative and gently resuspend. Repeat this process until the fixative supernatant is colorless.
6. Centrifuge at 150^for 5 min one last time and resuspend the pellet in 10 drops of fix if the pellet is large or 5 drops if the pellet is small. The cell/fix mixture should be slightly cloudy.
7. Place two drops of cell/fix mixture on opposing ends of a clean slide. Add one more drop of fix on top of the cells and let air-dry.
1. To detect positive amplification, use two template cover slips for DOP-PCR in situ. One will be used as a positive control by incorporating rhodamine-6-dUTP and the other will be used for microdissection and subsequent probe paint.
2. The reaction mixture has a 50-pL total volume and consists of 25 U Thermo Sequenase DNA Polymerase, 5 pL of Thermo Sequenase reaction buffer, 200 pM of each dATP, dTTP, dCTP, and dGTP, and 4 pM of DOP primer (5'-CCGACTCGAGNNNNNNATGTGG-3'). Control DOP-PCR in situ experiments included 40 pM tetramethylrhodamine-6-dUTP added to the reaction in addition to the other components. Place each reaction drop onto unfrosted microscope slides.
3. Carefully invert the template cover slip onto the slide with the reaction mixture so that the cells are immersed in solution.
4. Seal the edges with rubber cement. Carefully examine the seal for air bubbles after drying. Additional rubber cement should be administered if bubbles have formed (see Note 5).
5. Place the slides on a thermal cycler and without a heated lid use the following thermal profile; 95°C for 10 min, 8 cycles at 94°C for 1 min, 30°C for 5 min, and a ramp of 0.1°C/s up to 65°C for 5 min, 12 cycles at 94°C for 1 min, 56°C for 5 min, and 72°C for 5 min, followed by 72°C for 5 min and hold at 4°C until the slides can be removed (see Notes 6-8).
6. Carefully remove the cover slips with a razorblade or other sharp edge, and soak them in a 4X SSC, 0.1% triton X-100 solution for 5 min at room temperature.
7. Mount the fluorochrome labeled cover slip onto a microscope slide using the mounting medium (approx 10 pL for a 22 x 22-mm cover slip).
8. Blot away excess medium with a clean tissue.
9. Metaphase spreads can be visualized using a Zeiss Axioskop (Carl Zeiss, Inc) and images captured by a Vysis QUIPS Imaging Analysis System (Vysis).
10. PCR is considered successful if all choromosomes in a metaphase spread are labeled with fluorochrome.
1. Because of the sensitivity of whole-genome amplification, reaction set-up should be performed in a sterile hood. Sterile sleeves should be worn at all times, and great care must be taken to avoid contamination.
2. Template cover slips or slides must be denatured before amplification.
3. Denature the cover slips in 70% formamide, 2X SSC for 5 min at 70°C. Remove and run through the ethanol series: 70%, 85%, and 100% for 2 min each.
Whole-genome amplification is performed by mixing 10 ||L of 2 mM dNTP, 8 |L of 1X phi buffer mix, 2 |L of 4 mMDTT, 2 |L of 2 mM random hexamers, and 1.0 | L of phi29 DNA Polymerase per reaction.
Transfer the 21-|L reaction mixture onto the template cover slip and cover with a clean microscope slide.
Seal the edges with rubber cement. Carefully examine the seal for air bubbles. After drying air bubbles may have formed. Additional rubber cement should be administered if air bubbles are present.
Incubate on a thermal cycler, without a heated lid, at 30°C for the desired amount of time (4 h may be sufficient for subsequent microdissection, but amplification may occur for as long as 16 h. A larger reaction volume may be necessary for 16-h incubation owing to evaporation). Heat-denature the polymerase at 65°C for 10 min. Lower temperature to 4°C to chill.
Carefully remove the cover slip and set aside for subsequent microdissection. Microdissection
Place the glass pipet in the micropipet puller and pull needles to the desired tip width (see Note 9).
Place template cover slip face up onto the stage of the inverted microscope (see Note 10).
Place needle in the micromanipulator at angle of less than 45°. Needles may be broken by the cover slip if their resting angle is too steep. If the angle is too steep, it is hard to pick up dissected fragments; too low, and it is hard to cut fragments. Move the needle tip directly to the center of the field of view; approx 1 cm above the stage and template cover slip.
Lower the focal plane slightly, then lower the needle until it is in focus. Continue this process until the needle is just above the cover slip surface. This is preferable to lowering the needle and then focusing on it, which will generally result in the needle hitting the cover slip and breaking. Once the needle is just above the cover slip, rotate the objectives to the x100 lens.
At this point, the micromanipulator should be switched from coarse to fine motion. This process varies depending on the brand of micromanipulator, but if such a dual control system is present, the fine control makes movement at x100 much easier.
The needle should now be lowered slowly until it ceases moving down and moves slightly to the left. At this point, it should be moved to the left with the left-adjusting control of the micromanipulator, to scrape the chromosome (In a typical set-up, the needle will be coming from the right, pointing to the left.) Select your target, and orient it so that the needle will cut in the appropriate direction. In a scooping motion slowly move the tip of the needle down and at an angle under the cut fragment, or the whole chromosome or bacteria, carefully trying to get below the object. When the needle is located underneath the object, continue moving it laterally while slowly moving it up and away from the stage. The proper motion is a bit like an airplane taking off. Once the fragment is on the needle and off the surface, it is very unlikely to come off of the needle.
9. When the needle tip is a safe working distance from the stage (approx 1 cm), carefully remove the needle from the micromanipulator.
10. Carefully break off the tip of the needle into a dry, sterile 0.2-mL thin-walled microfuge tube for subsequent reactions.
11. Add the PCR master mix to the wall of the tube after the fragment(s) are collected, cap the tube and centrifuge to get everything to the bottom of the tube, and thermocycle as described in Subheading 3.4., step 5.
1. Polymerase choice is important; one must use a polymerase that is reasonably unimpeded by chromosome structure. We find that Thermo Sequenase works well, but there are others that we have not used, which may work as well or better. Experimentation may be useful.
2. A fluorescent nucleotide is unnecessary to the processes of DOP-PCR in situ and whole-genome amplification but is useful as a control to assess efficacy of the reaction. Typically, it is used in parallel with an unlabeled reaction; the products of the unlabeled reaction are used for subsequent amplifications.
3. The choice of polymerase is equally important for WGA. The ^29 bacteriophage DNA polymerase has an extremely high processivity and will produce large quantities of accurate product. It is, however, not thermostable, and must not be present during any denaturation steps.
4. This time can be varied according to many circumstances. The longer the col-cemid is left on the cells, the more condensed the metaphase chromosomes will become, which is advantageous for microdissecting whole chromosomes because it is easier to pick up condensed chromosomes. For isolating bands, it is much easier to use less condensed chromosomes. Colcemid times also depend on the fraction of mitotics desired and the division time of the individual cells. Chinese hamster ovary cells double in approx 12 h, half the time of human cells. To obtain a vary high fraction of mitotic human cells, we frequently leave the colcemid on the cultures for as long as 30 h.
5. Any gap in the rubber cement will allow the mix on the slide to evaporate. It is critical to have a well-sealed cover slip. Equally important is removing all air bubbles from between the slide and cover slip before applying the rubber cement, as they will expand and force out the liquid during heating. Gently lifting and lowering one corner of the cover slip with a razor blade or similar object will help remove the bubbles.
6. A flat block, dedicated to slides, is not necessary. Simply covering a normal microcentrifuge tube-capable block with aluminum foil is more than sufficient.
7. Humidity in the chamber is reasonably important because allowing the rubber cement to dry out can result in the PCR mix evaporating away during the reaction. It can occasionally be difficult to tell if this has happened by inspecting the slide, but very high backgrounds and spotty results are indicative of evaporation.
8. A primary issue with cycling PRINS and PCR in situ is product drift; if too many cycles are performed, fluorescently labeled product can leave the target locus and drift elsewhere. It is necessary to experiment to find the minimum number of cycles necessary to locate the desired locus, and not to perform more cycles than are absolutely necessary.
9. A handy needle holder consists of modeling clay flattened out in the bottom of an uncoated tissue culture plate; stick the unpointed end of each needle in the modeling clay and point straight up. The settings on the needle puller must be determined empirically, and depend on the type and thickness of capillary tube chosen, the puller itself, and the target to be isolated. For whole chromosomes and bacteria, a thicker needle is better. For chromosome bands or microchromosomes, a finer point is necessary.
10. This is a significant issue. It is impossible to tell on which side of the cover slip cells are without trying to scrape them with the needle. We have found that writing the word "cells" backward on the cover slip's noncell side works well. When the cover slip is held cell-side up, the word can be read. This will keep the ink off of the side on which reactions will take place.
This work was performed under the auspices of the US Department of Energy by University of California, Lawrence Livermore National Laboratory under Contract W-7405-Eng-48.
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