The heterogeneity of skeletal structures is one obstacle in studying the gene expression in developing cartilage and bone. This protocol provides a method for precise isolation of adjacent but different tissues. We optimize the laser capture microdissection for cartilage and the bone using cresyl violet staining to visualize these tissues for precise collection by maintaining high RNA integrity for subsequent analysis.
Other tissues can be isolated if cresyl violet provides sufficient distinction of target tissue and the RNA quality remains high after our processing method. Such tissues include craniofacial sutures and brain. Cryosectioning is the most demanding skill in this protocol.
Ensure that the tissue is dissected and embedded rapidly to maintain the highest integrity of RNA. To begin this procedure, dissect the embryo or tissue of interest. Embed the sample in a disposable embedding mold with optimal cutting temperature compound, and adjust the orientation of the specimen with a tip or needle.
Rapidly freeze the samples in a dry ice and methyl-2-butene bath. Next, prepare a lidded container with dry ice by placing a sheet of aluminum foil on the dry ice for temporary storage of dissection sample slides during cryosectioning. Transfer the fresh frozen specimen into the bucket of dry ice.
Place a layer of optimal cutting temperature compound onto a cryostat specimen holder, and immediately place the specimen on top of the optimal cutting temperature compound with gentle pressure. When the optimal cutting temperature compound is completely frozen, fasten the holder with the specimen into the cryostat cutting arm. Leave the sample in the cryostat for 15 minutes to equilibrate it to the cutting temperature.
Then, section the tissue at a thickness of 12 micrometers and collect the slices on PEN membrane slides. Align consecutive sections on the membrane. Once the slide is completed, allow the sections to dry for a few minutes, and then transfer the slide onto the foil in the dry ice container until all the required slides are collected.
Store the slides at minus 80 degrees Celsius in a slide box. To begin, prepare three labeled 15 milliliter centrifuge tubes. Add 45 milliliters of 80%ethanol to each tube.
Place three tubes on ice. Place a tube containing 45 milliliters of 95%ethanol and a tube containing 100%ethanol on ice. Next, add 45 milliliters of xylene to a 50 milliliter centrifuge tube at room temperature.
Thaw a PEN membrane slide briefly by placing it against a gloved hand, and wash it twice for 30 seconds in each of the tubes containing 80%ethanol with agitation to remove the optimal cutting temperature compound. Then, lay the slide on a sheet of aluminum foil. Pipette 8 milliliters of 1%cresyl violet and 50%ethanol onto the slide and stain for 30 seconds.
Wash the slide in the third tube containing 80%ethanol for 30 seconds. Dehydrate the slide by passing it through the tube containing 95%ethanol for 30 seconds, the tube containing 100%ethanol for 30 seconds, and finally, the tube containing xylene for 30 seconds. After this, place the slides on a delicate task wiper for five minutes to let the xylene drain and allow the sections to dry.
First, turn on the laser capture microdissection microscope, laser, and computer. Log on to the computer and start the software. When the green light is on, press the red button to activate the laser.
When the light turns red, the laser is ready to use. Insert the cap of a 5 milliliter PCR tube into the collector. Pipette 50 microliters of extraction buffer into the cap and insert the collection device into the microscope.
In the change collector device window, click on the move to reference point button to move the collector to the reference point. Adjust the focus to clearly view the reference point. Next, click the left unload button in the toolbar to lower the slide holder.
Place the slide into the holder with the tissue section facing downwards, and insert the holder back into the stage. Click continue in the change specimen window. To set up the laser parameters, select the control option of the laser menu or click on the laser icon.
Adjust the following parameters as desired, power, which is the power of the laser, aperture, which is the width of the laser, and speed, which is the cutting speed in draw and cut mode. Start with a 5X objective to find the areas of interest, and switch to the objective that best shows the area of interest. Choose the tube for collection by clicking the mark at the collector, then choose move and cut or draw and cut.
Once the cut is completed, the target tissue with PEN membrane will fall into the cap of the collection tube by gravity. Repeat this selection and cutting process to pull multiple areas of interest if needed. After this, unload the collector and carefully close the PCR tube.
Place the microdissected tissues on dry ice. Thaw the microdissected tissues at room temperature, then centrifuge briefly, and incubate the samples at 42 degrees Celsius for 30 minutes. After this, spin the lysis buffer down into the 5 milliliter PCR tube.
Perform DNase treatment and RNA extraction using an RNA isolation kit, following the manufacturer's instructions. In this study, an optimized laser capture microdissection of cartilage and bone is performed, highlighting the use of cresyl violet staining in a rapid procedure to visualize cartilage and bone for precise tissue collection while maintaining high RNA integrity for subsequent analysis. Observation of the slides shows that all cartilages examined are stained magenta, and all mineralized tissues are stained brown or black.
Both cartilage and bone are easily distinguished from other tissues at multiple anatomical sites. Meckel's Cartilage, condylar cartilage, and the mandibular bone regions are selected and isolated by laser capture microdissection. To obtain sufficient RNA for sequencing, pull 10 regions of Meckel's Cartilage, 10 regions of condylar cartilage, or four regions of mandibular bone into three individual collection tubes respectively.
RNA is then extracted, and the total RNA is analyzed using a bioanalyzer. This laser capture microdissection protocol is used on various tissues at different developmental stages, and the RNA integrity number measurements indicate high RNA quality. Differential gene expression analysis is then performed.
There are 4, 006 genes significantly and differently expressed between the mandibular bone and Meckel's Cartilage. Genes specific to osteoblasts or osteocytes are more highly expressed in the mandibular bone while chondrocyte specific genes are more highly expressed in Meckel's Cartilage. In addition, osteoclast markers are more highly expressed in the mandibular bone, indicating successful isolation of targeted tissues.
Following this procedure, gene expression analysis, including qPCR and RNA-seq, can be performed. These methods can analyze gene expression of the transcriptome in specific tissues or regions of interest within heterogeneous tissues.
