The overall goal of this procedure is to collect total RNA from discreet brain nuclei in a repeatable, consistent, cost efficient and RNAs free manner. This is accomplished by first preparing an RN free work environment and removing the whole brain from a decapitated mouse. Next, a micrometer operated tissue chopper is used to cut uniformly sized serial tissue sections.
Then brain regions of interest are micro dissected using a set diameter tissue core while maintaining tissue in an RNA free medium environment. Finally, total RNA is isolated from the excised brain nuclei. Ultimately, results can be obtained that show sufficient total RNA yield and quality for downstream CDNA library preparation and transcriptome profiling through spectra photometric or fluoro metric assays.
The main advantage of this technique over existing methods like gross anatomical or laser capture microscopy assisted brain region excision, is that multiple discrete brain regions of interest can be collected from an individual mouse brain in a repeatable and cost sufficient manner. This method can help answer key questions in the neuroscience field, such as whether distinct transcript expression profiles within brain regions associated with no neuronal circuits may be correlated with certain behavioral traits, environmental responses, and or disease state. To prepare for microdissection, supplement 100 milliliters of Earl's balanced salt solution or EBSS with 0.44 grams of sodium bicarbonate and 0.884 grams of glucose.
Treat the EBSS with 0.1 milliliters of DEPC in an autoclavable bottle or flask with a screw top for at least 12 hours. At 37 degrees Celsius. Place five and three quarter inches glass pipettes into a one liter glass beaker and place artists'brushes with bristles facing upward into a 100 milliliter glass graduated cylinder.
Add sufficient DEPC treated water solution to cover the pipettes and brushes. Cover the beaker and graduated cylinder with aluminum foil and incubate for at least 12 hours at 37 degrees Celsius after the 12 hour incubation with DEPC, heat, all DEPC treated materials to a temperature greater than 100 degrees Celsius for at least 15 minutes.Cool. The EBSS at room temperature prior to storage at four degrees Celsius.
Pour the DD H2O out from the beaker and graduated cylinder and continue to incubate them at greater than 100 degrees Celsius until the materials are dry. Remove them from the incubator, cool through room temperature and store for later use. On the day of the experiment, treat all work surfaces to remove RNAs contamination place and maintain the EBSS in a water bath set at 40 degrees Celsius to buffer and oxygenate.
The EBSS infiltrate it with 5%CO2 and 95%oxygen by placing gas tubing into an RNA free pasture pipette. And inserting the pipette into the EBSS cover the opening of the EBSS container. Set up a tissue slicer for serial sectioning of the brain tissue.
Treat the top and bottom surfaces of the tissue slicer. To remove RNAs contamination, attach a rectangular piece of watman Number four, filter paper to the plexiglass surface of the tissue chopper. Attach a double-edged razor blade to the chopper arm.
Set the micrometer to zero and adjust the minimum height of the chopper arm to allow the razor blade to cut the filter paper, but not the plexiglass plate. Gather all additional materials needed for the micro dissection, including RNA free 60 by 15 millimeter cell culture dishes, tissue cos 200 microliter RNA free micro pipette tips, curved scissors, dry ice, and tubes for tissue collection. Place the tubes on dry ice after decapitating a mouse using a rodent guillotine and removing the whole brain with both hind brain and optic nerves intact.
Place the brain into the top of a 60 by 15 millimeter RNAs free cell culture dish. Then transfer it to the tissue sampling work surface. Use an RNAs free pipette to conservatively moisten the filter paper attached to the tissue chopper with EBSS.
Wet the brain tissue with EBSS. Transfer the brain to the tissue chopper and position it with the ventral surface resting on the filter paper. The midline perpendicular to the chopper blade and the rostral edge of the brain abutting the blade.
Use A-D-E-P-C treated artist's brush to hold the brain stationary by applying gentle pressure to the dorsal surface of the brain while holding the brain stationary. Lift the blade holder lever about four to five centimeters above the dorsal tissue surface and hold it in this position. Remove the brush from the brain surface and dial 750 micrometers.
Using the micrometer. Allow the lever to drop chopping the tissue and creating a 750 micrometer coronal section while the chopper blade remains stationary. Use a rolling motion with the artist's brush to gently swipe the tissue section away from the blade surface.
Immediately place the tissue section into the oxygenated EBSS in the Petri dish. This slice is now ready for micro dissection. Repeat the slicing procedure until serial brain sections have been obtained across the entire rost codal plane to carry out micro dissection.
First, check the lighting to ensure that sample illumination will not be obstructed. Adjust the position and flow of the O2 CO2 mix to maintain a buffered oxygenated environment for the tissue. During the procedure, identify the coronal brain section that includes the region of interest and using an artist's brush, orient it flat on the bottom of the Petri dish.
Select the tissue core with the appropriate diameter. Hold the coronal tissue section in place with the artist's brush and bring the cutting tip of the tissue core down over the surface of the tissue section. While applying pressure, press the tissue core tip through the tissue section and down firmly onto the bottom of the Petri dish.
Use a rolling circular motion to ensure that the region of interest has been dissociated from the surrounding tissue. Carefully lift the tissue core away from the tissue section and liberate the micro punch allowing it to float in the EBSS Using an artist's brush. Immediately deposit the tissue micro punch into a minus 80 degrees Celsius Stable storage tube kept on dry ice.
The tissue should readily adhere to the side of the tube. Return the micros sample containing tube to the dry ice. Repeat this microdissection for each brain region of interest.
Once the microdissection is complete, record the respective label or barcodes for each sample containing tube. Place the tissue samples into a minus 80 degrees Celsius freezer until RNA extraction to extract RNA from the tissue samples. First, prepare an RNA free workspace.
Make a sufficient amount of magnetic bead mix and lysis binding solution as described within the total RNA isolation kit manual. Remove the tissue samples from the minus 80 degrees Celsius freezer and place the tubes directly onto dry ice. Add 100 microliters of the lysis binding solution to a tube of sample.
Attach a nuclease free 0.5 milliliter tube size pestle to the pellet mixer and homogenize the tissue using a low retention nuclease free pipette tip. Transfer the homogenized sample into a well on a round bottom 96 well tissue culture plate using the same type of tip immediately. Add 60 microliters of 100%isopropanol to the sample.
Add 20 microliters of the bead mix to the sample and mix by pipetting up and down three to four times. Follow the total RNA isolation kit protocol Steps to complete the total RNA extraction. Finally, measure the purity and concentration of resulting total RNA total RNA Data from micro punches of various brain regions are shown in this table.
RNA samples alluded with 30 microliters of evolution Buffer provided sufficient total RNA for use with commercially available CD NA preparation products for RNA sequencing applications. Additionally, the RNA yield and two 60 to two 80 ratios are single values obtained from respective microdissection samples to demonstrate readings obtained from each brain region and they confirm high sample purity Once mastered, this technique can be done in eight to 10 minutes if it is performed properly. While attempting this procedure, it's important to remember to minimize the risk of RNA contamination of work surfaces or tissue samples by only using RNA free or dsy treated materials.
