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 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.
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'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.
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
2. Frozen Section Preparation for Laser Microdissection
3. H&E Map Slide Staining Protocol and Morphological Review
4. Polyethylene Terephthalate (PET) Membrane Slides Processing and Staining Protocol
5. Laser Microdissection with UV Laser
6. Lysis of Microdissected TM Tissue
7. RNA Isolation and Analysis of Quality
8. Analysis
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 extremely low quantity of cDNA from the LCM samples digital PCR was used, which has been proven to be more reproducible with less material50. The expression of MYOC and ACTA2 was normalized using the HSP90a1 housekeeping gene, then each tissue was compared to that of the whole eye. These results show that MYOC (Figure 7A) and ACTA2 (Figure 7B) are highly expressed in the TM samples, confirming the successful isolation of high quality RNA from the highly enriched TM samples for downstream RNA analysis. As proof of concept, this technique was applied to the TM-isolated RNA prepared from WT mice and from a strain of mice with an elevated IOP phenotype and analyzed by microarray. This analysis identified many genes implicated in glaucoma that are differentially expressed between the TM of WT mice and those of the mice with the glaucoma phenotype (Figure 7C).
Figure 1: Placement of frozen tissue block in the cryostat. (A) O.C.T mounting medium is applied to the cryostat mounting chunk. (B) Frozen specimen block is evenly placed on the mounting chuck. Please click here to view a larger version of this figure.
Figure 2: Sectioning procedure of frozen eye tissue in the cryostat. (A) Preparation of cutting surface to reach eye tissue. (B) Trimming O.C.T. of the frozen block before collecting sections for laser microdissection. Please click here to view a larger version of this figure.
Figure 3: Preparation of frozen sections in the cryostat. (A) 6 serial 8 µm thick sections are lined up in the cryostat. (B) Sections are simultaneously mounted on the PET membrane slide. Please click here to view a larger version of this figure.
Figure 4: Screenshot of laser microdissection software highlighting important features. Please click here to view a larger version of this figure.
Figure 5: Location of trabecular meshwork and UV laser parameter settings for proper isolation of trabecular meshwork. (A) Low (4X) magnification of a single section on the PET membrane highlighting the trabecular meshwork for laser microdissection. (B) High (40X) magnification of trabecular meshwork and adjacent tissues. (C) First partial circle cuts of the TM near the scleral region with the cap in the up position. (D) Final cuts in the free space that complete the circle around the TM with the cap in the down position. (E) TM removed from the section and (F) attached to the cap. (G) Multiple cut sections cut from the same PET membrane not overlapping adhered to the cap. S (Sclera), SC (Schlemm's Canal), TM (Trabecular Meshwork), AC (Anterior Chamber). Please click here to view a larger version of this figure.
Figure 6: Total RNA yield and quality estimation obtained from microdissected trabecular meshwork tissue. (A) Total RNA from 80 sections of trabecular meshwork (approximated area size was 0.8mm2). (B) Total RNA from 20 sections of trabecular meshwork (approximated area size was 0.2mm2). (C) Total RNA from remaining eye tissue after laser microdissection. RIN, RNA integrity number; bp, base pairs. Please click here to view a larger version of this figure.
Figure 7: Downstream RNA analysis of LCM isolated trabecular meshwork. Quantitative digital PCR analysis of TM-associated genes in the TM isolated by LCM. The TM-associated genes (A) MYOC (B) ACTA2 highly expressed in the LCM captured TM samples compared to other eye tissues. (C) Heatmap generated from data obtained from microarray analysis of trabecular meshwork RNA isolated from WT mice and KO mice with an elevated IOP phenotype. Please click here to view a larger version of this figure.
Figure 8: Comparison of tissue preparation before and after optimization. Eye sections cut and placed on PET membrane slides either with (A) the standard 95% ethanol dehydration preparation (B) or the optimized 75% ethanol dehydration preparation. Please click here to view a larger version of this figure.
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 are not yet fully understood21,22,23,24,25. Genetic mouse models can provide a powerful tool for studying the molecular mechanisms of glaucoma. However, the small size of mouse TM tissue represents a formidable challenge for isolation of highly-enriched TM from mouse eyes and high quality RNA for gene expression analysis. LCM is a valuable technique for isolating a single or low number of cells and can be used to study gene expression in tissue samples enriched for specific cell types. In this study, a robust and reproducible LCM method is described to isolate highly enriched TM and high quality RNA that can be used to study the regulation of gene expression and cell signaling in the TM. In addition, the described LCM method can also be applied to other tissues within the eye.
LCM technology and a high attention to detail for optimization of tissue handling played a vital role in the successful development of this method. The methodology of tissue preservation and isolation is a key component in isolating high quality RNA for gene expression profiling. LCM requires a very high attention to detail in dissecting, cryosectioning, fixation, staining, dehydration, and time duration of laser microdissection to be successful in obtaining quality RNA51. Tissue section thickness can be increased; however, as the thickness increases so does the UV laser power needed. Increased laser power results in a wider laser path that can cause RNA degradation. It was found that the laser path with 8 µm sections was thin and provided enough tissue for downstream RNA analysis. Figure 8 shows how optimization of the tissue preparation can drastically alter the ability to laser dissect the TM from the eye. The optimization of this step in the protocol (as described in section 4) yielded superior ocular-tissue immobilization and complete removal of O.C.T. (Figure 8B) by simply using 75% instead of 95% ethanol (Figure 8A) to dehydrate the tissue followed by incubation in RNase-free water to remove mounting media. Further dehydration and staining is achieved by using alcohol-based staining reagents. In addition, it was found that alcohol-based staining reagents yielded superior results relative to aqueous staining reagents (water-based cresyl violet or hematoxylin) and further helps preserve RNA integrity52. Overall, processing ocular tissues using the optimized protocol with alcohol-based staining reagents yielded consistent ocular-tissue sections that were fully immobilized on the PET membrane slide.
Our goal for this study was to develop a robust and reliable method for isolating high quality mRNA from the TM of mouse eyes for downstream expression analysis in order to obtain insights into the molecular mechanisms that underlie elevated IOP and glaucoma. As shown in the heatmap in Figure 7C, the described protocol of isolating TM by LCM provides a very reliable method to isolate RNA from TM and study differences in gene expression in TM from wild type and mutant mice. The method described herein will allow investigators to perform molecular analysis on a tissue central to the pathogenesis of glaucoma in an in vivo system that is relatively easy and reliable and can be applied to gene expression analysis.
The authors have nothing to disclose.
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 |