Osteocyte gene expression analysis in mouse bone: optimization of a laser-assisted microdissection protocol

Abstract Among bone cells, osteocytes are the most abundant, but also the most challenging to study because they are located inside a dense mineralized matrix. Due to their involvement in bone homeostasis, diverse tools are needed to understand their roles in bone physiology and pathology. This work was aimed at establishing a laser-assisted microdissection protocol to isolate osteocytes and analyze their gene expressions. The goal was to overcome the limitations of the technique currently most used: RNA extraction from the whole bone. To perform laser microdissection and subsequent gene expression analysis, the five main steps of the protocol have been adapted for the bone tissue. After testing many parameters, we found that the best options were (1) take unfixed snap-frozen tissue, (2) cryosection with a supported tape system to improve the tissue morphology if necessary, (3) microdissect regions of interest, and (4) recover the bone pieces by catapulting, if feasible, or by gravity. Finally, RNA extraction (5) was the most efficient with a precipitation method and allowed quantifying the expression of well described osteocyte genes (Gja1/Cx43, Phex, Pdpn, Dmp1, Sost). This work describes two protocols optimized for femur and calvaria and gives an overview of the many optimization options that one could try when facing difficulties with laser microdissection.


Protocol 1 (femur) STEP 1: Bone sample preparation
The protocol is designed based on experiments with adult mouse bones.The main goal of this step is to prepare the bone samples to allow for efficient cryosectioning and to preserve the RNA integrity.
1. Prepare molds for cryopreservation (1 mold/femur): add OCT (Optimal Cutting Temperature gel, TFM-C); avoid to introduce air bubbles.Let the molds at room temperature.
2. Prepare a container with liquid nitrogen.16.Adjust parameters depending on the section quality: objective 20x; laser beam energy: 78%; focus: 75%; speed: from 10% to 30%.17.Draw on the screen the areas that will be microdissected.We recommend to draw and dissect the areas one by one to adjust the number of cutting cycles if necessary.
18.The dissected areas will fall on the coverslip.It is possible to focus on the coverslip to confirm the presence of the bone pieces that were cut.19.Within 2 hours a total surface of 2.5 ± 0.1 mm 2 can be dissected.We have not tested the RNA quality with a longer period of time.

Protocol 2 (calvaria) STEP 1: Bone sample preparation
The protocol is designed based on experiments with adult mouse bones.The main goal of this step is to prepare the bone samples to allow for efficient cryosectioning and to preserve the RNA integrity.
2. Prepare a container of isopentane placed in liquid nitrogen.

3.
Euthanize mice by cervical dislocation and immediately dissect the femurs.There is no need to remove the periosteum or flush the bone marrow.4. Embed bones in OCT and immediately dive the molds in liquid nitrogen.5.When all the dissections are done, transfer the molds at -80 °C for storage.STEP 2: Cryosectioning 6. Adjust the cryostat temperature to -27 °C ± 1 °C and install a blade specifically designed for hard tissue.Clean all the instruments with RNase AWAY™.7. When the temperature is reached in the cryostat chamber, place the molds containing the bone samples in the chamber and let them equilibrate in temperature for 15 min.Position one sample on the tissue holder with OCT. 8. Adjust the tissue thickness to 5 µm and transfer the section to a PET FrameSlide; if possible, put 3 sections on the same FrameSlide.The sections should be as flat as possible to allow the laser beam to cut efficiently.Keep the slide in the cryostat chamber or continue to step 3. STEP 3: Dehydration and staining of sections 9. Prepare containers with ice-cold ethanol 50% (x2), 75% (x2), 95% (x2), and 100% (x2).10.Dive the FrameSlide successively in ethanol 95%, ethanol 75%, and ethanol 50% for 40 s in each bath.11.Put the FrameSlide on a paper and add a drop of the solution of Cresyl Violet (1% in ethanol 50%).Let stain for 10 s. 12. Transfer immediately the FrameSlide in the bath of ethanol 50 %, and proceed to dehydration with the successive baths of ethanol 75%, and twice 100% for 40 s in each bath.13.Let the FrameSlide dry completely at room temperature.STEP 4: Microdissection and collection of osteocyte-containing bone pieces 14.Immobilize a coverslip (0.13-0.16 mm) below the FrameSlide with a tape.The bone section should be between the PET membrane and the coverslip.15.Place this assembly on the slide holder of the P.A.L.M MicroBeam device with the coverslip below.

20 . 21 .
After 2 hours, take carefully the FrameSlide, remove the tape that immobilizes the coverslip and add lysis solution; use this solution to transfer the bone pieces into a microtube.Store the microtube containing the bone pieces at -80 °C until RNA extraction.STEP 5: Tissue lysis and RNA extraction 22. Use the MasterPure Complete DNA & RNA Purification Kit, and follow the protocol recommended for tissue samples.23.Add 1 µL of Proteinase K to the Tissue and Cell Lysis Solution.Homogenize the bone pieces in this solution.24.Incubate at 65 °C for 15 min, and regularly homogenize by vortexing after 5 and 10 min.Let the solution a few minutes on ice.25.Add 150 µL of the solution "MPC Protein Precipitation Reagent", and vortex for 10 s. 26.Centrifuge for 10 min at 4 °C at ≥ 10,000 g to discard the debris.Transfer the supernatant into a new microtube.27.Add 500 µL of isopropanol.Invert the microtube 30-40 times.28.Centrifuge for 10 min at 4 °C at ≥ 10,000 g to pellet the nucleic acids.29.Remove isopropanol without touching the nucleic acid pellet.30.Rinse with 70% ethanol and centrifuge.Remove all of the residual ethanol.Repeat this step.31.Resuspend the total nucleic acids in 15 μL of TE Buffer.Store at -80°C until the reverse transcriptase step.
3. Euthanize mice by cervical dislocation and immediately dissect the calvaria.Depending on the region of interest (ROI) in the calvaria, it may be interesting to cut the calvaria in the middle or near the ROI (with a scalpel blade) to present this region on an edge of the mold and to cut directly in the ROI after only a few sections.4. Embed bones in SCEM embedding medium and immediately dive the molds in liquid nitrogen.5.When all the dissections are done, transfer the molds at -80 °C for storage.STEP 2: Cryosectioning 6. Adjust the cryostat temperature to -27 °C ± 1 °C and install a blade specifically designed for hard tissue.Clean all the instruments with RNase AWAY™.7. When the temperature is reached in the cryostat chamber, place the molds containing the bone samples in the chamber and let them equilibrate in temperature for 15 min.Position one sample on the tissue holder with SCEM.8. Cut a few pieces of Kawamoto LMD film with scissors.The adhering part is in the center, framed by two non-adhering regions.