Name: laser capture microdissection of highly pure trabecular meshwork from mouse eyes for gene expression analysis
Uploaded: 2018-06-03T13:00:00
Description: Watch this Scientific Journal Video about Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis at JoVE.com

The National Institute of Environmental Health Sciences (NIEHS) Animal Care and Usage Committee (ACUC) approved all methodology of this study under the NIEHS Animal Study Proposal IIDL 05-46.
1. Optimal Tissue Collection for Laser Microdissection
<ol>
	<li>Obtain 2 to 3-month-old mice, male or female C57BL/6. Euthanize with CO2 for a minimum of 1 min or until respiration has ceased. Remove the animal from the cage and assure death by either cervical dislocation, decapitation, or t...

The TM plays a vital role in actively maintaining homeostatic IOP and its dysfunction is widely accepted as the main causative factor for hypertensive glaucoma1,2,19. A number of single nucleotide polymorphisms in several genes identified by GWAS analysis have been linked to increased glaucoma risk and increased resistance to AH outflow facility at the TM; however, the precise molecular mechanisms that give rise to this disease ...

Glaucoma is a group of diseases characterized by optic neuropathy and retinopathy that ultimately leads to irreversible blindness1,2. It is estimated that by 2020 over 70 million people worldwide will be living with some form of the disease3,4,5,6,7. Primary open angle glaucoma (POAG), the most prevalent type of glaucoma, is characterized by a decrease in aqueous humor (AH) outflow leading to increased intraocular pressure (IOP)8,9,10,11,12,13,143,15,16,17,18. Left untreated, chronically elevated IOP leads to progressive and irreversible damage to the retina and optic nerve head causing radial blindness1,2,19. All current methods for slowing the progression of glaucoma focus on reducing IOP, either by decreasing the rate of production of AH by the ciliary body or enhancing it&#39;s outflow1,8,9,10,11,12,13,14. The trabecular meshwork (TM) plays a vital role in actively regulating the primary AH outflow pathway and its improper function is a causative factor for hypertensive glaucoma1,2,19. However, the molecular mechanisms associated with TM dysfunction and how it regulates AH drainage are not yet fully understood and is currently a major focus of glaucoma research1,2,19,20. While several genome-wide association studies (GWAS) have linked a number of genes to glaucoma and increased resistance to AH outflow facility at the TM, the exact molecular mechanisms that lead to disease are not yet fully understood21,22,23,24,25.
Animal models have greatly enhanced our current knowledge of disease progression in glaucoma (extensively reviewed in3,15,16,26,27,28,29,30,31,32,33). Several pioneering methods have been developed to study the TM34,35,36 and these methods have been widely used to advance our current understanding of normal and diseased tissue. One area that has not been extensively explored is the use of genetically modified mouse models to study the molecular mechanisms of TM failure. Transgenic knock-in and knock-out mouse studies of TM associated genes, such as Myocilin (Myoc)37,38 and Cyp1b139, have been the primary tools for studying the molecular mechanisms of TM function. Understandably, the small size of the TM in mice represents a serious hurdle that must be overcome in order to begin to study this tissue. Mouse models represent a powerful tool for studying the genetics and molecular mechanisms of disease, while advances in LCM technologies provide the necessary tools to empower the study of the smallest and most delicate tissues, including the TM.
In this report, a robust and reproducible method is described for the LCM of highly enriched TM from mouse eyes along with subsequent RNA isolation, and amplification for downstream expression analysis. Similar methods have been used successfully in mice to isolate other types of eye tissues40,41,42,43,44, the methodology reported herein can be applied to other discrete tissues of the eye to study RNA, microRNA, DNA, and proteins. Importantly, this technique enables the use of genetically modified mice to better understand the molecular pathogenesis of TM impairment in glaucoma and ocular disease3,15,16,17,18,26,31,45,46. The ability to isolate the TM of mouse eyes by LCM will be a useful technique in obtaining further insights into the molecular mechanisms of several ocular diseases.

<table>
	<tbody>
		<tr>
			<td>ACTA2 ddPCR Primers (dMmuCPE5117282)</td>
			<td>BioRad</td>
			<td>10031252</td>
			<td>FAM</td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2946-90004</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent RNA 6000 Pico kit</td>
			<td>Agilent Technologies</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>BioRad QX200 Droplet Digital PCR System</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Small Paint Brush</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Charged Glass Microscope Slide</td>
			<td>Thermo scientific</td>
			<td>4951PLUS-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cresyl Violet Acetate</td>
			<td>Sigma Aldrich</td>
			<td>C5042</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y dye</td>
			<td>Thermo scientific</td>
			<td>71204</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td>Curved and Serrated tip (preferred tip size: 0.5 x 0.4 mm)</td>
		</tr>
		<tr>
			<td>HemaCen</td>
			<td>American MasterTech</td>
			<td>STHEM30</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>Applied Biosystems</td>
			<td>4368814</td>
			<td></td>
		</tr>
		<tr>
			<td>Hsp90a ddPCR Primers(dMmuCPE5097465)</td>
			<td>BioRad</td>
			<td>10031255</td>
			<td>VEX</td>
		</tr>
		<tr>
			<td>Leica CM1850 Cryostat</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GS filter unit</td>
			<td>EMD Millipore</td>
			<td>SLGS033SB</td>
			<td>0.22 &#181;m</td>
		</tr>
		<tr>
			<td>MMI CellCut UV Cutting Model</td>
			<td>Molecular Machines &#38; Industries</td>
			<td></td>
			<td>LCM intrument</td>
		</tr>
		<tr>
			<td>MMI CellTools Software</td>
			<td>Molecular Machines &#38; Industries</td>
			<td>50202</td>
			<td>LCM software</td>
		</tr>
		<tr>
			<td>Sample Tube for Laser Capture Microdisssection</td>
			<td>ASEE Products</td>
			<td>ST-LMD-M-500</td>
			<td>Isolation Cap Tube/Manufactured by Microdissect GmBH in Germany and distrubted by ASEE Products</td>
		</tr>
		<tr>
			<td>Sample Tube for Laser Capture Microdisssection (Alternative)</td>
			<td>Molecular Machines &#38; Industries</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>modified Harris Hematoxylin</td>
			<td>Thermo scientific</td>
			<td>7211</td>
			<td>FAM</td>
		</tr>
		<tr>
			<td>MYOC ddPCR Primers (dMmuCPE5095712)</td>
			<td>BioRad</td>
			<td>10031252</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Memebrane Slides, RNase Free</td>
			<td>ASEE Products</td>
			<td>FS-LMD-M-50r</td>
			<td>Polyethylene terephthalate (PET) membrane/Manufactured by Microdissect GmBH in Germany and distrubted by ASEE Products</td>
		</tr>
		<tr>
			<td>Memebrane Slides, RNase Free (Alternative)</td>
			<td>Molecular Machines &#38; Industries</td>
			<td>50102</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapid Fix</td>
			<td>Thermo scientific</td>
			<td>6764212</td>
			<td>H&#38;E staining</td>
		</tr>
		<tr>
			<td>RLT Buffer</td>
			<td>Qiagen</td>
			<td>79216</td>
			<td>lysis bufffer used for LCM samples</td>
		</tr>
		<tr>
			<td>RNAseZap</td>
			<td>Sigma</td>
			<td>R2020</td>
			<td>RNase decontamination solution</td>
		</tr>
		<tr>
			<td>Protect RNA RNAse Inhibitor</td>
			<td>Sigma Aldrich</td>
			<td>R7397</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Micro Kit</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td>RNA isolation kit</td>
		</tr>
		<tr>
			<td>SMART-Seq v4 Ultra Low Input RNA Kit</td>
			<td>Takara Clontech</td>
			<td>634888</td>
			<td>low input RNA to cDNA kit for LCM samples</td>
		</tr>
		<tr>
			<td>SuperMix (no dUTP)</td>
			<td>BioRad</td>
			<td>1863023</td>
			<td>digital PCR master mix</td>
		</tr>
		<tr>
			<td>Tissue-Tek Cryomold (25mm x 20mm x5mm)</td>
			<td>Sakura</td>
			<td>4557</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek O.C.T. Compound</td>
			<td>Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Stratalinker UV Crosslinker</td>
			<td>Stratagene</td>
			<td>400075</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Macron</td>
			<td>8668</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser capture microdissection of highly pure trabecular meshwork from mouse eyes for gene expression analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

LCM collected RNA from the TM and ciliary body from 4 different mice was isolated in order to be able to analyze gene expression and compare the expression with that in whole eye, sclera, iris, retina, cornea, and lens isolated from three separate mice. TM expressing genes, MYOC48 and ACTA249 were analyzed in all the collected tissues to confirm that the isolated TM samples were indeed highly enriched in TM. Due to the extr...

Watch this Scientific Journal Video about Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis at JoVE.com

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

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