Name: laser capture microdissection of mouse embryonic cartilage and bone for gene expression analysis
Uploaded: 2019-12-18T13:00:00
Description: Watch this Scientific Journal Video about Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis at JoVE.com

This work was supported by the National Institute of Dental and Craniofacial Research (R01DE022988) and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (P01HD078233). The authors thank the Biorepository and Pathology Core for access to the Leica LMD 6500 platform at the Icahn School of Medicine at Mount Sinai.

Tissues from mice were obtained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and study protocols were approved by the Institutional Animal Care and Use Committee at the Icahn School of Medicine at Mount Sinai.
1. Preparation of Fresh Frozen Specimen
<ol>
	<li>Dissect the embryo or tissue of interest. Embed the sample in a disposable embedding mold with optimal cutting temperature (OCT) compound. Adjust the orientation of the specimen ...

LCM enables the isolation of enriched or homogenous cell populations from heterogeneous tissues. Its advantages include rapid and precise capture of cells in their in vivo context, while potential disadvantages include it being time consuming, expensive, and limited by the need for the user to recognize distinct subpopulations within a specified sample30. This protocol provides details of LCM of mouse embryonic cartilage and bone, highlighting the use of cresyl violet staining in a rapid ...

The musculoskeletal system is a multicomponent system composed of muscle, connective tissue, tendon, ligament, cartilage, and bone, innervated by nerves and vascularized by blood vessels1. The skeletal tissues develop with increasing cellular heterogeneity and structural complexity. Cartilage and bone develop from the same osteochondroprogenitor lineage and are highly related. Embryonic cartilage and bone develop in association with muscles, nerves, blood vessels, and undifferentiated mesenchyme. Cartilage may also be surrounded by bone, such as Meckel's cartilage and condylar cartilage within the mandibular bone. These tissues are anatomically associated and interact with each other through extracellular signals during development. In the study of gene expression in the development of cartilage and bone, one obstacle is the heterogeneity of skeletal structures composed of multiple tissue types. Precise isolation of the specific tissue of interest is key for successful transcriptional analysis.
Laser capture microdissection (LCM) is a powerful tool to isolate cell types or regions of interest within heterogeneous tissues, and is reproducible and is sensitive to the single cell level2. It can precisely target and capture cells of interest for a wide range of downstream assays in transcriptomics, genomics, and proteomics3,4. The quality of the isolated RNA, DNA, or protein can be assessed with a bioanalyzer or equivalent platform. For example, RNA quality is indicated by the RNA integrity number (RIN)5.
Here, we provide a protocol for the rapid staining and isolation of cartilage and bone by LCM from fresh frozen tissues. We use the mouse embryo to demonstrate that this protocol yields high quality RNA for subsequent transcriptomic analysis, such as RNA sequencing (RNA-seq).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Methylbutane</td>
			<td>ThermoFisher Scientific</td>
			<td>O3551-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent</td>
			<td>G2939BA</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>ThermoFisher Scientific</td>
			<td>339653</td>
			<td>Conical sterile polypropylene centrifuge tubes, 50 mL</td>
		</tr>
		<tr>
			<td>Cresyl violet acetate</td>
			<td>Sigma-Aldrich</td>
			<td>C5042</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica Biosystems</td>
			<td>CM3050 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate task wiper</td>
			<td>ThermoFisher Scientific</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable embedding mold</td>
			<td>ThermoFisher Scientific</td>
			<td>1220</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>Invitrogen</td>
			<td>10977-015</td>
			<td>DNase/RNase-Free</td>
		</tr>
		<tr>
			<td>Ethanol, absolute (200 proof)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP2818</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Glass PEN membrane slide</td>
			<td>Leica Microsystems</td>
			<td>11505158</td>
			<td></td>
		</tr>
		<tr>
			<td>LCM system</td>
			<td>Leica Microsystems</td>
			<td>Leica LMD6500</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass</td>
			<td>ThermoFisher Scientific</td>
			<td>12-545FP</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>ThermoFisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT compound</td>
			<td>Electron Microscopy Sciences</td>
			<td>102094-106</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tube with flat cap, 0.5 mL</td>
			<td>Axygen</td>
			<td>PCR-05-C</td>
			<td>LCM collection tubes</td>
		</tr>
		<tr>
			<td>Permanent mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA isolation kit</td>
			<td>ThermoFisher Scientific</td>
			<td>KIT0204</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase decontamination agent</td>
			<td>Sigma-Aldrich</td>
			<td>R2020</td>
			<td>RNase decontamination agent for cleaning surfaces</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma-Aldrich</td>
			<td>214736</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser capture microdissection of mouse embryonic cartilage and bone for gene expression analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

Coronal sections of fresh frozen mouse tissues at E16.5 were used to demonstrate the isolation and collection of Meckel&#39;s cartilage (MC), condylar cartilage, and mandibular bone by LCM. Mouse embryos at E16.5 were dissected and embedded in cryogenic molds with OCT compound. Samples in molds were rapidly frozen in a dry ice and methyl-2-butane bath and stored at -80 &#176;C.
To demonstrate cresyl violet staining of cartilage and bone, cryosectioning in the coronal plane was performed and sa...

Watch this Scientific Journal Video about Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis at JoVE.com

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

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