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Method Article
This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.
Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (≥8) as needed for transcriptional profiling experiments.
Laser capture microdissection (LCM) is a valuable research tool that allows the separation of pathologically relevant cells for subsequent molecularly driven evaluation. The use of molecular analyses in these heterogeneous tissue specimens and the correlation with pathological and clinical data is a necessary step in evaluating the translational significance of biological research1. When analyzing gene expression data from RNA, the use of frozen tissue sections is highly recommended as it allows for excellent quality of RNA as well as maximized quantity2. It has been well established that high quality and quantity of RNA are essential for meaningful data from RNA sequencing3. However, when using RNA from fresh frozen post-mortem tissue for LCM, RNA degradation is a major challenge, as it occurs immediately upon death and its extent is mediated by various factors associated with the tissue collection method4,5. Furthermore, RNA degradation is exacerbated when staining techniques are needed to recognize histologic details and cell identification. Specialized staining techniques, such as hematoxylin & eosin, Nissl stain, immunofluorescence and immunohistochemistry are helpful in differentiating cells from surrounding stroma but have been shown to degrade RNA and alter transcript expression profiles6. Therefore, our laboratory has created a stainless protocol specifically designed to preserve RNA in post-mortem human cerebellum for the purposes of RNA sequencing after LCM isolation of Purkinje neurons.
In processing fresh frozen tissue for LCM, the fixation method can variably affect both RNA and tissue integrity. Formalin fixation is standard for morphological preservation, but causes cross-linking that may fragment RNA and interfere with RNA amplification7. Ethanol fixation is a better alternative for RNA isolation, as it is a coagulative fixative that does not induce cross-linking1. To enhance the visualization of tissue morphology, xylene is the best choice, as it removes lipids from the tissue. However, there are known limitations when utilizing xylene in LCM, as the tissues can dry out and become brittle causing tissue fragmentation upon laser capture7. Xylene is also a volatile toxin and must be handled properly in a fume hood. Nevertheless, xylene has been shown to enhance tissue visualization while also preserving RNA integrity8. Therefore, our protocol centers around the use of 70% ethanol fixation and ethanol dehydration, followed by xylene incubation for morphological clarity.
It is important to note the different types of laser-based microdissection systems, as they have been shown to differ in speed, precision, and RNA quality. The infrared (IR) laser capture microdissection and the ultraviolet (UV) laser microbeam microdissection systems were both novel LCM platforms that emerged almost concurrently8. The IR-LCM system employs a "contact system" using a transparent thermoplastic film placed directly on the tissue section, and cells of interest selectively adhere to the film by focused pulses from an IR laser. Alternatively, the UV-LCM system is a "non-contact system" whereby a focused laser beam cuts away the cells or regions of interest in the tissue; depending on the configuration in two currently available commercial platforms that use either an inverted or upright microscope design, tissue is acquired into a collection device by a laser-induced pressure wave that catapults it against gravity or tissue is collected by gravity, respectively. Significant advantages of the UV-LCM system include faster cell acquisition, contamination-free collection with the non-contact approach and more precise dissection due to a much smaller laser beam diameter9. This protocol was specifically designed for a UV-LCM system and has not been tested in an IR-LCM system. In either UV-LCM system design, when accumulating cells into the collection cap, the use of a coverslip that enhances cellular clarity during microscopy is not suitable, as the cells would be unable to enter into the collection cap. Therefore, to enhance tissue visualization, we tested the use of Opaque collection caps, which are designed to act as a coverslip for microscopic visualization in UV-LCM systems, against liquid filled collection caps. Liquid filled collection caps can be challenging, as the liquid is subject to evaporation and must be replaced frequently while working at the microscope. Time becomes an important factor for RNA stability, as the captured tissue dissolves immediately7.
Our laboratory studies the postmortem neuropathologic changes in the cerebellum of patients with essential tremor (ET) and related neurodegenerative disorders of the cerebellum. We have demonstrated morphologic changes centered on Purkinje cells that distinguish ET cases versus controls, including a reduced number of Purkinje cells, increased dendritic regression and a variety of axonal changes, leading us to postulate that Purkinje cell degeneration is a core biologic feature in ET pathogenesis10,11,12,13. Transcriptional profiling has been used in many neurologic diseases to explore the underlying molecular basis of degenerative cellular changes. However, transcriptional profiles from heterogeneous samples, such as brain tissue regions, can effectually mask the expression of low abundance transcripts and/or diminish the detection of molecular changes that occur only in a small population of affected cells, such as Purkinje cells in the cerebellar cortex. For instance, Purkinje cells are vastly outnumbered by the abundant granule cells in the cerebellar cortex by approximately 1:3000; thus, to effectively target their transcriptome requires specific isolation of these neurons. The cerebellar cortex is delineated by distinct layers that differ in cell density and cell size. This cellular architecture is ideal to visualize in a fresh-frozen tissue sample without dye-containing staining reagents. In theory, this protocol could also be applied to other tissue types that have similar distinctive tissue organization.
This protocol was designed to work specifically for Purkinje cell visualization in the human post-mortem cerebellum. Numerous protocols exist for the fixation, staining visualization and RNA preservation of many types of tissues for the purposes of both IR- and UV-LCM. When contemplating an experimental design for UV-LCM, individuals should tailor their protocol to best fit the needs and requirements of starting and ending materials. Here, we combine many aspects of different LCM protocols to provide an enhanced method for visualizing Purkinje cells in the post-mortem human cerebellum without the need of dye containing staining reagents to prepare high-quality RNA for transcriptome sequencing.
All human samples utilized in this protocol have been obtained with informed consent and have been approved by the Internal Review Board (IRB) at Columbia University and Yale University.
NOTE: The entirety of this protocol should follow strict RNA handling guidelines, whereby a gloved hand is always used, all surfaces are cleaned with an RNase decontaminator and all working materials are RNA/DNA/Nuclease free.
1. Test RNA Integrity of Tissue prior to Starting LCM
NOTE: Testing RNA quality can be done by many methods. Ensure that RNA from the entire section is tested to provide a representative RNA integrity number (RIN) for the sample.
2. Prepare prior to Starting Sectioning for LCM
3. Prepare Fixation Solutions with RNase Free Water and High-Quality Ethanol
NOTE: All solutions are prepared fresh for every experiment. Ensure that the slide holder cleaning procedure from Step 1.1 is completed. All solutions are prepared in slide holders.
4. Prepare Cryostat for Tissue Sectioning
5. Sectioning Tissue
6. Fixing Tissue/Stain-less Visualization
7. Optional — Slide Storage
8. Thawing Stored Slides for Use
9. Laser Capture Microdissection of Purkinje Cells:
NOTE: This section of the protocol will only discuss specifics related to capturing Purkinje cells for subsequent RNA sequencing. This section assumes that the user is familiar with the UV laser capture microscope and the affiliated software.
10. RNA Collection post LCM
This protocol details the steps for preparing fresh frozen post-mortem human brain tissue for UV-LCM. Thorough specifications and annotations are given for cryostat cutting, as it can be difficult to cut fresh frozen brain tissue with a high degree of precision. The most important point to consider is that fresh frozen tissue is extremely cold and requires a significant amount of time to acclimate to the warmer temperature of a cryostat. This step cannot be rushed, and it cannot be overst...
The protocol presented here is specifically modified to be a stainless approach in visualizing morphologically distinct tissues for UV-LCM. This method is designed to maximize RNA integrity for subsequent direct RNA sequencing, while maintaining an enhanced level of tissue visualization. The ability to distinguish different cell types for capture, to create the purest population of cells possible, is essential for understanding different molecular profiles in human tissues15. Within the context of...
The authors have nothing to disclose.
The authors would like to acknowledge The New York Brain Bank, Dr. Jean Paul Vonsattel and Dr. Etty Cortés, for their assistance in cutting and preserving the frozen human brain tissue samples for these experiments. The authors would like to acknowledge the individuals who generously donated their brain for the continuing research into Essential Tremor. Dr. Faust and Dr. Martuscello would like to acknowledge Columbia University Department of Pathology and Cell Biology for their continued support and core research space. The authors would like to acknowledge NIH R01 NS088257 Louis/Faust) for research funding for this project. LCM images were performed in the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by NIH grant #P30 CA013696 (National Cancer Institute). The authors would like to acknowledge Theresa Swayne and Laura Munteanu for their continued assistance with specialized microscopy.
Name | Company | Catalog Number | Comments |
MembraneSlide NF 1.0 PEN | Zeiss | 415190-9081-000 | Membrane slides for tissue. We do not recommend using glass slides. |
AdhesiveCap 500 Opaque | Zeiss | 415190-9201-000 | Opaque caps are used to enhance visualization on inverted scopes only |
RNase Away | Molecular BioProducts | 7005-11 | Other RNase decontamination products are also suitable. Use to clean all surfaces prior to work. |
200 Proof Ethanol | Decon Laboratories | 2701 | If using alternative Ethanol, ensure high quality. Essential for tissue fixation. |
Xylenes (Certified ACS) | Fisher Scientific | X5P-1GAL | If using alternative xylene, ensure high quality. Essential for tissue visualization. |
UltraPure Distilled Water | Invitrogen | 10977-015 | Other RNA/DNA/Nuclease free water also suitable. Utilized in ethanol dilution only. |
Slide-Fix Slide Jars | Evergreen | 240-5440-G8K | Any slide holder/jar is also suitable. Must be cleaned prior to use. Used for fixation following sectioning. |
Anti-Roll Plate, Assy. 70mm 100um | Leica Biosystems | 14041933980 | Anti-roll plate type is determined by type of cryostat and attachment location on stage. All cryostats have differing requirements. |
Diethyl pyrocarbonate (DEPC) | Sigma Aldrich | D5758-50mL | DEPC from any vendor will do. Follow directions for water treatment. |
Kimwipes | Fisher Scientific | 06-666A | Kimwipes can be ordered from any vendor and used at any size. Kimwipes are to be used to clean microscope and cryostat. |
Tissue-Plus O.C.T. Compound | Fisher Scientific | 23-730-571 | Any brand of OCT is acceptable. No tissue will be embedded in the OCT, it is strickly to attach tissue to 'chuck'. |
Edge-Rite Low-Profile Microtome Blades | Thermo Scientific | 4280L | Blade type is determined by type of cryostat. All cryostats have differing requirements. |
Leica Microsystems 3P 25 + 30MM CRYOSTAT CHUCKS | Fisher Scientific | NC0558768 | Chuck type is determined by type of cryostat. All cryostats have different requirements. Either size is suitable. |
RNeasy Micro Kit (50) | Qiagen | 74004 | Required for RNA extraction post LCM. RLT Lysis buffer essential to gather captured cells from opaque cap. |
RNeasy Mini Kit (50) | Qiagen | 74104 | Required for RNA extraction of section preps, which are performed prior to LCM to test RNA integrity of starting tissues. |
RNase-Free DNase Set | Qiagen | 79254 | Dnase will remove any DNA to allow for a pure RNA population. Ensure Dnase is made fresh. |
2-Mercaptoethanol | Sigma Aldrich | M6250-100ML | Purchased volume at user discretion. Necessary for addition to RLT buffer for cell lysis. Prevents RNase activity. |
Globe Scientific Lot Certified Graduated Microcentrifuge Tube (2 mL) | Fisher Scientific | 22-010-092 | Any 2 mL tube that is RNase free, DNase free, human DNA free, and Pyrogen free will do. |
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