A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

This protocol is significant, because it utilizes a stain-free visualization method instead of dye-based visualization on post-mortem human brain tissue, which helps preserve RNA integrity. The preservation of RNA is a big advantage in this protocol, as it is useful, not only in post-mortem human brain tissue, but also other tissue types, with high RNase activity. To begin, remove the tissue from the freezer and place it on dry ice for transport to the cryostat.

Place the clean brushes, the chuck, and the tissue into the cryostat for at least 20 minutes, to allow them to reach the optimal temperature. For cryostats with dual chambers and specimen-holding temperatures, set the object temperature to minus 16 degrees Celsius and the chamber temperature to minus 18 degrees Celsius. Set the cryostat's section thickness to 14 micrometers and the trim thickness to 30 micrometers.

Next, clean the stage with a one-to-one mixture of RNase decontaminator and 70%ethanol, to prevent freezing. Obtain a new, disposable cryostat blade and clean it with RNase decontaminator. Place the cleaned blade in the blade holder on the stage.

Then, clean the anti-roll plate with the RNase decontaminator. Attach the cleaned plate to the stage and wait at least 20 minutes for the blade and anti-roll plate to come down to the temperature of the cryostat. First, cut a small piece of tissue for sectioning, making sure that the section is approximately 95%cerebellar cortex.

Return the remaining tissue either to dry ice, or a to a freezer, at minus 80 degrees Celsius. Next, begin slowly adding OCT on top of the chuck with a slow, circular motion. Build up layers of OTC until there is a mount approximately three millimeters high covering the chuck.

When the OCT is partially frozen, but still has some liquid in the center, place the tissues on top of the mount. Let the tissue sit on top of the OCT for one to two minutes until it is completely frozen. Then, transfer the chuck with the frozen OCT and tissue into the cryostat cutting arm.

Adjust the tissue so that it is flush with the cutting blade and let the tissue sit in the cutting arm for 20 minutes to adjust to the new temperature. The most critical step is allowing the tissue to acclimate in the cryostat cutting arm for as long as necessary. If tissue shreds, purkinje cell visualization will be very difficult.

I recommend using smaller, thinner pieces of tissue, to allow the tissue to acclimate faster. After this, slowly move the tissue closer to the blade. Once the tissue has reached the blade, begin the trimming process.

Trim the tissue two to three times, until the cortex layers are visible. Next, place and align the anti-roll plate just above the cryostat blade. Cut four to six sections that are 14 micrometers thick, noting that properly cut sections will be flat under the anti-roll plate.

Align the cut sections horizontally across the cryostat stage. Angle a slide to pick up all of the tissue pieces simultaneously. Immediately after sectioning the tissue, place the slide in the slide holder with 70%ethanol on ice for two minutes.

Transfer the slide to a slide holder with 95%ethanol on ice for 45 seconds. Next, place the slide in the slide holder with 100%ethanol at room temperature for two minutes. Dip the slide three times into the slide holder containing xylene number one at room temperature.

Then, place the slide in the slide holder with xylene two at room temperature for five minutes. Stand the slides up in a clean fume hood and let them air dry for at least 30 minutes. If storing the slides, place each one into a separate 50 milliliter tube and place the tubes into a freezer at minus 80 degrees Celsius for up to seven days.

First, use RNase decontaminator to clean the microscope stage and the cap collection arm. With gloved hands, place the slide on the microscope and a 500 microliter opaque cap in the collection arm. At a low magnification, align the opaque cap over the cerebellar tissue, insuring that the cap covers the entire area that is visualized in the eye piece.

Use a five times to 10 times objective to visualize the cerebellar layers. Place the cursor over the section where molecular layer and granual cell layer intersect. Move to a 40 times magnification and visualize the purkinje cells.

Then, begin capturing them. To begin RNA collection after the microdissection, add 50 microliters of cell lysis buffer to the opaque cap with the cap facing up. Carefully close the tube over the cap and proceed with the collection as outlined in the text protocol.

In this protocol, fresh, frozen, post-mortem, human brain tissue is prepared for UV laser capture microdissection. Following cryostat's sectioning in the allotted drawing time, the cellular layers of the cerebellum are easily visible with five times and 10 times objective lenses. As seen here, proper xylene incubation causes the tissue to become darker and better delineates cellular layers than does ethanol alone.

When cutting at the laser capture microscope, the 40 times objective lens is required to ensure capturing only purkinje cells and not the surrounding tissue. As seen here, proper incubation in xylene produces a high quality morphologically intact image when compared to ethanol fixation alone. The cover slip ability of an opaque cap is then tested while other protocols utilize a liquid-filled cap, these caps can reduce tissue visualization and cause the resulting image to be granular and iridescent under the microscope.

However, visualization through an opaque cap results in the smoothened tissue appearance that is softer and sharper in form. Representative images of excised purkinje cells at low power and at high power show precise removal of just the purkinje cell body. After this, high quality Rnase is obtained for subsequent RNA sequencing.

Six representative samples underwent RNA extraction and were resuspended in 14 microliters of Rnase-free water and all six samples produced high quality RNA with RNA integrity numbers equal to or greater than eight. The most important thing to remember is that tissue quality is everything. Even cutting and slide placement will make or break this protocol.

Following this procedure, any method that investigates RNA or DNA could be performed, specifically we probe our RNA for differential gene expression, but other questions regarding gene regulation could also be investigated. This method could be applied to any tissue type that has specific delineated morphology that does not require the use of antigen or dye-specific reagents for identification of cells of interest. We hope that this technique will help us understand the genetics of essential tremor and other diseases that have a tremor phenotype.

The most hazardous chemical used in this protocol is xylene. It should be handled properly in a fume hood, disposed of properly, and slides should be allowed to dry adequately until used in an open space. Additionally, when working with human samples, biohazardous waste management, and safety should be utilized.