This protocol uses laser microdissection and RNA sequencing to analyze the differential mRNA expression of intratumor heterogeneity, such as areas of accumulation of mesenchymal cells or areas of tumor invasion. This method has been optimized to preserve good quality tissue histology and RNA integrity to facilitate the acquisition of high quality laser microdissection samples. This method is sensitive and highly reproducible.
It can be utilized to study tumor morphology and heterogeneity in various tumor models. Laser microdissection of frozen brain tumor tissue is a cost effective, reliable technique for the isolation of discrete anatomical areas of cell populations from tumor tissues to study their molecular profiles. LMD can be utilized to gain in-depth mechanistic knowledge about the molecular events that take place during tumor progression, and could reveal novel therapeutic targets.
An in vivo bioluminescence of the mouse tumor burden reaches a signal between one times 10 to the six and one times 10 to the 7th photons. Flush oxygenated tyrode solution through the circulatory system of the euthanized tumor-bearing mouse until the liver and lungs have been completely cleared of blood. Continue perfusing the animal with 30%sucrose solution dissolved in tyrode solution for an additional 15 minutes.
To evaluate the success of the perfusion, confirm that the neck, tail and legs are rigid at the end of the circulation. Then harvest the brain according to standard protocols and store the brain overnight in fresh 30%sucrose solution. To prepare for the cryopreservation, fill a jar with cold isopentane-2-methylbutane and place the jar into a container filled with liquid nitrogen.
While the solvent is chilling, blot the brain dry on a piece of filter paper and place the brain into a labeled Cryomold. Carefully at approximately five milliliters of optimal temperature cutting medium to the center of the Cryomold and place the brain into the mold in the desired orientation. Using clean forceps, quickly place the Cryomold into the chilled isopentane-2-methylbutane for 30 to 40 seconds.
Once the cutting medium has solidified, transfer the Cryomold onto dry ice and wrap the Cryomold and snap frozen brain tissue in aluminum foil for minus 80 degrees Celsius storage. To acquire frozen brain tumor tissue sections, first set the temperature of a cryostat between minus 20 and minus 24 degrees Celsius, and place the sample block into the cryostat chamber for 30 to 60 minutes. While the sample is equilibrating, clean the chamber and knife holder with 100%ethanol and spray the section brushes with RNase cleaning solution.
When the sample is ready, remove the mold and use fresh cutting medium to attach the frozen tissue block to the cryostat's specimen disc. Place the block in the disc holder and align the block with the knife blade. Acquire 10 micrometer sections of the brain using one of the Rnase-free brushes to carefully flatten and uncurl the tissue pieces onto the cutting surface.
To mount the brain tissue sections onto the slides, smoothly press the positively charged side of a labeled RNase-free PEN glass slide onto the section and place the slide into a storage box inside the chamber. When all of the slides have been collected, place the storage box at minus 80 degrees Celsius. To dissolve the cutting medium before laser micro dissection, submerge the slides in 30-second sequential descending ethanol immersions followed by crystal violet solution staining for 20 seconds and five seconds in 0.5%eosin Y solution.
At the end of the eosin Y incubation, use filter paper to block the slides dry and dehydrate the slides in sequential ascending ethanol immersions. After the 60-second ethanol immersion, rinse the slides in a container of xylene for three minutes. Next, dry the slides on RNase-free surface at room temperature for 10 seconds before mounting the samples in mounting medium prepared with RNase-free water.
After 10 to 20 seconds, transfer the slides onto the microscope microdissection platform. For laser capture microdissection, turn on the power before turning on the laser. Next, turn on the microscope controller in the computer and start the laser capture microdissection software.
In the Microscope Control panel, select the 10 times magnification. Under Laser Control, set the laser parameters for tissue dissection and select a laser frequency of 120 hertz and a laser current of 100%For accurate laser microdissection, set the speed to 10 and set the aperture to 10 micrometers. Then set the power to 53.
Load the tissue collector that will capture the tissue following the section and click the second Unload button. Replace the empty collector with DNase and Rnase-free 0.5 milliliter PCR flathead tubes containing 30 microliters of lysis buffer per tube. Return the collector to the machine and click continue to proceed.
Load the processed specimen onto the microscope and click Unload in the laser microdissection software. Up the sample onto the slide holder and place the slide holder onto the stage. Click Continue to proceed and select Draw Cut in the Cut Shapes window.
Use the microscope controls to find the area of interest and draw an outline around the region of interest. Then select a destination collector tube and click Start Cut to proceed the tissue microdissection. When all of the areas of interest have been dissected, transfer the collector tubes from the holder onto dry ice until they're downstream processing.
To acquire tissues with a superior morphology and RNA integrity, various perfusion approaches were evaluated. To dissect the areas of interest, it is necessary to stain the tissues with innocuous dyes for RNA. The subsequent perfusion of tumor-bearing mice with tyrode solution and 30%sucrose followed by an overnight incubation in 30%sucrose results in the preservation of the morphology and RNA integrity of the mouse tissue.
Although paraformaldehyde tissue fixation results in a high quality tissue morphology, the RNA integrity is negatively impacted. Other approaches such as using a tyrode solution incubation, or tyrode solution plus 30%sucrose solution incubations do not affect the RNA quality, but a reduced resolution in the tissue morphology is observed. If the tissues are not mounted with mounting medium, the sections become dehydrated and the morphology deteriorates while mounting of the tissue samples with 15%mounting medium dissolved in water maintains a high quality tissue morphology.
Laser capture microdissection microscope imaging allows selection of the tumor tissue for dissection. This region of interest selection then allows dissection of areas of interest within each tissue sample for RNA integrity analysis. Maintaining a high quality tumor tissue morphology and RNA integrity is critical.
Tyrode solution and 30%sucrose perfusion with overnight preservation in 30%sucrose significantly improves tissue morphology for LMD. Quick lyoma fixation, staining and mounting with GAN solution is critical step to preserving RNA quality and preventing cracks from forming within the tissue. We recommend sectioning at least 2.5 times 10 to the 6th square micrometers total tumor per tissue area to obtain appropriate amounts of RNA for both RNA quality control and transcriptomic analysis.
LMD enables the analysis of the molecular signaling pathways that regulate glioma heterogeneity and invasion which could reveal novel potential targets for future translational development in preclinical glioma models.
