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

Summary

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.

Abstract

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.

Introduction

Glaucoma is a group of diseases characterized by optic neuropathy and retinopathy that ultimately leads to irreversible blindness^1,2. It is estimated that by 2020 over 70 million people worldwide will be living with some form of the disease^3,4,5,6,7. 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

Protocol

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

1. 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 thoracotomy.
2. Before dissection, ensure all dissection tools are clean and sterilized.
   NOTE: For the removal of the eyes curved scissor....

Results

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.......

Discussion

The TM plays a vital role in actively maintaining homeostatic IOP and its dysfunction is widely accepted as the main causative factor for hypertensive glaucoma^1,2,19. 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 .......

Materials

List of materials used in this article

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