Have you ever thought about harnessing the power of laser capture microdissection for analysis of gene expression in specific bone cells within their natural environment? Here, we describe a protocol that will enable you to obtain sufficient quantities of high quality RNA from cryosections of mouse bones. This technology is a great tool for examining changes in gene expression in specific bone cells in genetically engineered mouse models or in disease models.
The protocol described here is focused on mouse bones, but can be used to study in situ a gene expression in cells of any hard tissue in any species. Helping to demonstrate this procedure, will be Christiane Schueler, a technician from my laboratory. After euthanizing mice by exsanguination under general anesthesia, remove whole femurs rapidly.
Use a scalpel and paper towels to clean the femurs of the surrounding soft tissues. Next, pour optimal cutting temperature compound into the embedding mold and place the femurs into the bottom of the embedding molds. Snap freeze the samples in liquid nitrogen.
Once the samples are entirely frozen, wrap them in foil and transfer them on dry ice to a freezer. Store them at minus 80 degrees Celsius until ready for further processing. First, set the temperature in the cryostat to minus 19 degrees Celsius and the temperature of the block holder to minus 17 degrees Celsius.
Next, wipe down the interior of the cryostat with 70%ethanol. Place the disposable blade for hard tissues, the glass slides, and a fitting tool into the cryostat to cool, and keep them inside the cryostat for the duration of sectioning. Transfer the frozen tissue block on dry ice to the cryostat and allow it to equilibrate for at least 10 minutes.
Then, press the bottom of the embedding mold to push the OCT block out of the mold. Apply enough OCT medium to the block holder to adhere the block to it and wait until the OCT medium freezes completely. After this, place the block holder in the object holder and tighten it in place.
Adjust the blade position and trim the block in 15-micrometer cutting increments to remove the OCT covering the sample. Adjust the cryostat to generate eight-micrometer sections and cut two to three cryosections that will be discarded. Place adhesive film on the block and use the fitting tool to adhere the film to the block.
Now, make a cut slowly and at constant speed, while holding the section by the film. Place the film with the sample facing up on a pre-cooled glass slide on the cryobar within the cryostat to avoid thawing of the sample. Use tape to fix the film to the glass slide for easier staining.
In a fume hood, prepare the necessary solutions of ethanol, RNase-free water, and xylene on ice as outlined in the text protocol. Incubate the sections in 95%ethanol for 30 seconds. Then, carefully dip the sections in RNase-free water for 30 seconds to completely remove the OCT.
Next, dispense 50 microliters of commercial LCM frozen section stain onto the section and incubate at room temperature for 10 seconds. Drain the section by placing the edge of the slide on absorbent tissue paper. Rinse the section in 100%ethanol for 30 seconds to remove any excess stain.
Immerse the bone sections in a second tube containing 100%ethanol for 30 seconds and then, transfer them to 100%xylene for 30 seconds. Put adhesive film on a dry glass slide as a support, taking care to place the film as flat as possible. After this, place a PET membrane frame slide on the film and briefly press a gloved finger on the membrane to attach it to the film.
This sample will be sandwiched between the membrane and the adhesive film. The adhesive film should not be folded or wrinkled and there should be no air bubbles between the film and the membrane. Start by using surface decontaminant to clean the stage end cap holder.
Load the slide into the slide holder and the caps into the cap holder. Next, adjust the focus and acquire a slide overview with the 1.25x objective. Change to the 40x objective and adjust the focus.
Using the slide overview, choose the area of interest. Then, adjust the laser parameters as shown here, making sure to optimize these parameters for each objective. If the laser fails to cut the sample, increase the laser power.
Select osteoblast, osteocites, and bone lining cells in distal femoral cancellous or cortical bone based on morphologic criteria. Draw a line for the laser path further away from the target cells to minimize the damage by the UV laser. If the laser fails to cut the sample, apply the laser more than once or inspect the target for spots of incomplete cuts and use the move and cut option to cut the tissue in these spots.
Collect each cell type in a separate 0.5-milliliter tube cap. First, dispense 50 microliters of the lysis buffer containing beta mercaptoethanol into the cap of the collection tube. Lyse the sample by pippetting it up and down in the cap for one minute.
Next, spin down the lysate and add 00 microliters of the lysis buffer containing beta-mercaptoethanol to the tube. For each slide, prepare a labeled microcentrifuge tube with 350 microliters of the lysis buffer containing-beta mercaptoethanol. Use the sections remaining after the LCM to extract RNA.
Carefully separate the film from the membrane and lyse the sample by slowly pippetting the lysis buffer onto the section several times. Then, put the lysate samples on dry ice and store them at minus 80 degrees Celsius. When ready to continue, thaw the lysates at room temperature.
After this, transfer the lysates from LCM-harvested cells in the collection tubes to new 1.5-milliliter microcentrifuge tubes and extract RNA according to the manufacturer's instructions. In this study, an LCM protocol is developed to obtain sufficient quantity of high quality RNA for gene expression analysis in bone cells of mouse femurs. LCM is performed using an LCM system that uses gravity for sample collection.
The yield and integrity of isolated RNA was measured using microcapillary electrophoresis. The difference in RNA quality and quantity obtained using different lysis protocols can be seen here in the representative gel and electropherograms. When the sample is lysed by pippetting up and down in the cap for one minute, it is possible to isolate approximately 8.5 nanograms of RNA from one square millimeter of microdissected bone tissue.
The RIN value is 8.60. Alternatively, LCM is performed using an LCM system that uses a defocused laser pulse, which catapults the material into the overhanging adhesive cap. For fresh frozen bones, it is possible to isolate approximately 1.6 nanograms of RNA from one square millimeter of microdissected bone tissue.
The RIN value is one. In conclusion, the most important things to remember in this procedure are apply the standing protocol in the correct manner, prevent complete dryout of sections in the sandwich complex, use an appropriate LCM system, and do not exceed the time limits for harvesting the cells. Once you have isolated RNA from the captured cells, you can use the RNA for expression profiling, for example, by RNA seek.
Finally, we would like to remind you that xylene and beta-mercaptoethanol are hazardous substances that need to be handled under a hood. In addition, please take appropriate precautions when handling liquid nitrogen and also the cryotome blades.
