The authors would like to thank James Fosse for his excellent work on all videography and video processing; Michael Marti for his excellent work in the generation of digitized cattle images; Lilia Walther for her help with RNA extraction and Bioanalyzer runs; Mitch Palmer and Carly Kanipe for their helpful review and feedback on lymph node images; and the animal care and veterinary staff at the National Animal Disease Center for all of their hard work and assistance with animal husbandry and the preparation for necropsies.

The authors have no conflicts of interest to disclose. All research is funded with intramural funds from the U.S. Department of Agriculture, Agricultural Research Service. All references to specific products are provided for the purpose of experimental reproducibility and do not represent any endorsement of these products by the federal government.

The majority of transcriptomic studies and the associated protocols focus on mouse, rat, or post-mortem human samples. However, investigations in livestock and wildlife provide a wide range of opportunities for the characterization of the immune response to disease, both as applicable to veterinary medicine and, in regard to zoonotic diseases, to human public health. This protocol provided an outline of key considerations for high-integrity RNA extraction from tissues from large animals, such as cattle, bison, goats, and...

RNA-sequencing analysis of the transcriptome of lymph nodes provides the opportunity to characterize the immune response of animals to a variety of pathogens. While this methodology has been utilized extensively in mice, analyses have recently been expanding into larger mammals1,2. Livestock/large animal lymph nodes can be used to characterize host responses to an infection, not only for their use in vaccine or genetic susceptibility studies and for the identification of targets for drug development, but also as model systems for human studies on zoonotic diseases. For example, in the case of brucellosis (a zoonotic bacterial disease that impacts half a million people around the world each year), despite significantly increased costs, studies in sheep or goats are more relevant to the human infection and human vaccine development than laboratory animal models. Mouse infection models recapitulate the reticuloendothelial system infection but not the characteristic clinical signs3. 
In large animal experiments as compared to laboratory animal studies, the process of tissue harvesting necessarily involves a longer delay between the euthanasia and the tissue collection, which presents a potential challenge for the preservation of high-quality RNA. Intact RNA is essential for the generation of biologically relevant transcriptomic data. The generation of high-quality RNA from tissue samples is particularly critical for large animal pathogen studies conducted in containment facilities. Such studies are inherently more difficult to perform as they not only require approved facilities and highly trained personnel but also carry significant financial costs, which, depending on the work, can range from tens to hundreds of thousands of dollars. These types of studies also involve a cross-disciplinary collaboration and cross-disciplinary knowledge for their completion, adding to their complexity. Therefore, training on, development of, and adherence to a streamlined system for the sample collection and preservation provides significant benefits for downstream molecular studies of tissues from infected animals.
The collection of larger lymph nodes presents additional challenges for the tissue collection compared to the similar sampling of murine lymph nodes. The preparation for the sample excision necessitates a basic understanding of the anatomy of the lymph node, including the relevant internal structures. The structure of a lymph node is comprised&#160;of lymphoid lobules surrounded by sinuses filled with lymph. These structures are enclosed within a tough, fibrous capsule.4 A lymphoid lobule is the &#34;basic anatomical and functional unit of the lymph node&#34; and is composed of follicles, a deep cortical unit, and medullary cords and sinuses4 (Figure&#160;1A). B and T lymphocytes are home to the follicles and deep cortical units, respectively. These structures provide a 3D scaffold and facilitate the interaction between the lymphocytes and antigen or antigen presenting cells.
Grossly, follicles and deep cortical units can be identified on cut surface as they contain a denser reticular meshwork and appear darker than the sinuses, which are comprised of a more delicate reticular meshwork and appear lighter (Figure 1B). By convention, pathologists refer to the regions of the lymph nodes as the superficial cortex (follicles), the paracortex (deep cortical units) and the medulla (medullary cords and sinuses). A proper examination of all three regions has been deemed as best practice in routine pathological examination guidelines for lymph nodes5. Note that there is a considerable variation in the consistency, size, and color of lymph nodes, even within a single animal. As animals age, their lymph nodes will tend to decrease in size and become firmer than those of younger animals, typically due to an increase in their connective tissue and a reduction of the normal lymphoid structure6,7.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57195/57195fig1.jpg" /> 
Figure 1.&#160;Anatomy of the lymph node. (A) This cartoon image shows the anatomy of the lymph node, depicting key structures. (B)&#160;This still image shows a bovine lymph node cut in cross-section. The relevant structures/layers that are visible to the naked eye are highlighted. <a href="https://www.jove.com/files/ftp_upload/57195/57195fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Depending on the experimental question, different lymph nodes will be of interest for the collection and analysis. Peripheral lymph nodes are those located deep in the subcutaneous tissue. In cattle, peripheral or superficial lymph nodes often used in clinical and experimental practice include parotid, submandibular, retropharyngeal, prescapular, prefemoral (precrural) and superficial inguinal (supramammary in females, scrotal in males) (Figure 2). In Table 1, the properties of key superficial lymph nodes, as described in the cattle system8, are outlined. Below, some potential lymph node collection plans for infectious bacterial diseases of cattle are presented as a starting point for the investigation.
Brucella abortus/Brucella melitensis: Standard necropsies for B. abortus-infected cattle and B. melitensis-infected goats at the National Animal Disease Center recover supramammary, prescapular, and parotid lymph node tissue, both for the grinding for the bacterial enumeration and for the RNA preparation for the host RNA expression profiling. B. abortus can be regularly recovered in each of these lymph nodes in experimentally infected cattle9. The presence of bacteria in each of these lymph node types can be detected in B. melitensis-infected goats up to at least nine months post-infection using the RNA-based methodologies from our studies (Boggiatto et al., unpublished). Salmonella sp.: The prescapular, subiliac (prefemoral), and mesenteric lymph nodes have been useful during the profiling of cattle carcasses for a Salmonella prevalence10,11,12 and would be of potential interest for transcriptomic studies. E. coli O157:H7: Mesenteric lymph nodes (at the middle small intestine and distal small intestine locations) can be the sites of an occasional recovery of the bacteria in infected calves (but not in infected adult cattle)13. Leptospirosis (Leptospira sp.): A chronic persistence of the bacteria has been observed in the lymph nodes draining the mammary gland14. Mycobacterium bovis: In cattle, the bacteria have been recovered post-experimental infection from the mediastinal and tracheobronchial lymph nodes of calves15. Additionally, lymph node RNA has been utilized to examine large animal host responses to viruses, such as the porcine reproductive and respiratory syndrome virus2. Figure 2 depicts the location of a subset of these major lymph nodes in the cattle body.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57195/57195fig2.jpg" /> 
Figure 2: Cartoon depicting selected lymph node locations in Bos taurus. The numbered lymph nodes are annotated. <a href="https://www.jove.com/files/ftp_upload/57195/57195fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In this paper and the associated video, we present a protocol for the isolation of large animal lymph nodes for RNA studies, designed to be informative for molecular biologists involved in transcriptomic studies of large animal infections. First, we provide an overview of the isolation procedure for the lymph nodes, using sampling from bovine and bison tissues as examples. Paired with this demonstration, as displayed in the video, is a workflow for a reproducible tissue sampling for RNA isolation. Next, we describe important considerations for the processing of an infected lymph node, with a focus on safety, consistency, and RNA quality.
The preparation of RNA from the tissue with an acidified phenol-guanidine isothiocyanate reagent is based on the original method of Chomczynski and Sacchi16,17, with a purification over silica-based spin columns in the presence of chaotropic agents based on the original work of Vogelstein and Gillespie18. We also examine the potential for the recovery of RNA for transcriptomics from cattle lymph nodes preserved by alternative methods. Finally, we explore the impact of the time variable on the RNA quality in large animal necropsies, including a representative experiment depicting the effect of an increase in time between the euthanasia and the sampling on the recovered RNA profile from bison and bovine lymph nodes. This article will be useful not only to molecular biologists but also to veterinary researchers commencing transcriptomic studies.

<table><tbody><tr><td>RNA preservation solution (we used RNALater for all experiments)</td><td>ThermoFisher</td><td>AM7020</td><td></td></tr><tr><td>1.5 ml or 2 ml polypropylene microcentrifuge tubes&amp;nbsp;</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Disposable scalpels</td><td>Daigger Scientific</td><td>EF7281</td><td></td></tr><tr><td>Tissue forceps, rat tooth</td><td>Fisher Scientific</td><td>12-460-117</td><td>Other tissue forceps available including curved tip, tapered edge, etc. , depends on user preference</td></tr><tr><td>3 mm punch biopsy needles</td><td>Fisher Scientific</td><td>NC9949469</td><td></td></tr><tr><td>Sharps container (small and transportable for necropsy)</td><td>Stericycle</td><td>8900SA</td><td>1 qt. size shown here</td></tr><tr><td>Cutting boards or disposable trays</td><td>Fisher Scientific</td><td>09-002-24A</td><td>Available in a variety of sizes, depends on user preference</td></tr><tr><td>Personal protective equipment</td><td></td><td></td><td>Varies with pathogen (gloves, respirator masks, goggles, etc.)</td></tr><tr><td>Phenol-based RNA extraction reagent (we used TRIzol Reagent for all experiments)</td><td>ThermoFisher</td><td>15596026</td><td></td></tr><tr><td>Silica column-based RNA extraction kit (we used the PureLink RNA Mini kit for all experiments)</td><td>ThermoFisher</td><td>12183018A</td><td>Designed for up to 100 mg tissue</td></tr><tr><td>100% Ethanol (200 proof for molecular biology)</td><td>Sigma-Aldrich</td><td>E7023</td><td></td></tr><tr><td>Tissue homogenizer with enclosed homogenization tubes (we used the gentleMACS dissociator for all experiments)</td><td>Miltenyi Biotec</td><td>130-093-235</td><td></td></tr><tr><td>Agarose (General, for gel electrophoresis)</td><td>Sigma-Aldrich</td><td>A9539</td><td></td></tr><tr><td>1X TBE</td><td>Fisher Scientific</td><td>BP24301</td><td>Can also make from scratch in the laboratory</td></tr><tr><td>Deionized formamide</td><td>EMD Millipore</td><td>S4117</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma-Aldrich</td><td>L3771</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma-Aldrich</td><td>114391</td><td></td></tr><tr><td>Xylene cyanol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>X4126</td><td></td></tr><tr><td>EDTA (Ethylenediaminetetraacetic acid)</td><td>Sigma-Aldrich</td><td>EDS</td><td></td></tr><tr><td>UV-Vis Spectrophotometer (we used the NanoDrop Spectrophotometer)</td><td>ThermoFisher</td><td>ND-2000</td><td></td></tr><tr><td>Device for quantitative RNA assessment (we used the Bioanalyzer, with associated components and protocols)</td><td>Agilent</td><td>G2939BA</td><td></td></tr><tr><td>FFPE RNA extraction kit (we used the RecoverAll Total Nucleic Acid Isolation Kit for Formalin Fixed, Paraffin Embedded Tissue)</td><td>ThermoFisher</td><td>AM1975</td><td></td></tr><tr><td>Plastic spreader (L-shaped spreader)</td><td>Fisher Scientific</td><td>14-665-231</td><td>Only needed for sterility testing for samples from infected animals</td></tr><tr><td>Necropsy knives</td><td>Livestock Concepts</td><td>WI-0009209</td><td></td></tr></tbody></table>

collection and processing of lymph nodes from large animals for rna analysis: preparing for lymph node transcriptomic studies of large animal species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

The use of the considerations presented in this article (steps 1 - 4&#160;of the protocol) will aid in the recovery of RNA from large animal samples that is suitable for a downstream analysis in host gene expression studies. The RNA quality for downstream applications is assessed by multiple standard measures. For spectrophotometry, the A260/A280&#160;ratio provides a measure of the protein contamination, and the A260/A230&#160;ratio provides another means of purity assessment ...

Watch this Scientific Journal Video about Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species at JoVE.

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

collection-processing-lymph-nodes-from-large-animals-for-rna-analysis

Research

JoVE Journal

Immunology and Infection

26.1K Views.  U.S. Department of Agriculture. This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

Video: Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Mouse Adipose Tissue Collection and Processing for RNA Analysis

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and Western Blot, since it mostly contains lipids. RNA isolation from adipose tissue samples is also challenging as extra steps are required to avoid these lipids. Here, we present a procedure to collect three anatomically different white adipose tissues from mice, to process these samples and perform RNA isolation. We further describe the synthesis of cDNA and gene expression experiments using real-time PCR. The hereby described protocol allows the reduction of contamination from the animal's hair and blood on fat pads as well as cross-contamination between different fat pads during tissue collection. It has also been optimized to ensure adequate quantity and quality of the RNA extracted. This protocol can be widely applied to any mouse model where adipose tissue samples are required for routine experiments such as real-time PCR but is not intended for isolation from primary adipocytes cell culture.

The purpose of this paper is to present a step-by-step procedure to collect different white adipose tissues from mice, process the fat samples and extract RNA.

Compared to other tissues, white adipose tissue has a considerably less RNA and protein content for downstream applications such as real-time PCR and ...

Biochemistry

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence of disseminated tumor cells in patients with different types of cancer. The detection of these LN-residing tumor cells is an important biomarker associated with poor prognosis and adjuvant therapy decisions. Recent mouse models have indicated that LN-residing tumor cells could be a substantial source of malignant cells for distant metastases. The ability to quantify the adhesivity of tumor cells to LN parenchyma is a critical gauge in experimental research that focuses on the identification of genes or signaling pathways relevant for lymphatic/metastatic dissemination. Because LNs are complex 3D structures with a variety of appearances and compositions in tissue sections depending on the plane of section, their matrices are difficult to replicate experimentally in vitro in a fully controlled way. Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to LN cryosections. Using serial sections of the same LN, we adapt the classic method developed by Brodt to use nonradioactive labels and directly count the number of adhering tumor cells per LN surface area. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the cell-binding affinity to LN parenchyma, which is suggestive evidence of molecular alterations in the affinity binding of integrins to their correlate LN-ligands.

Here, we describe a simple and inexpensive method that allows the quantification of adhesive tumor cells to lymph node (LN) cryosections. LN-adherent tumor cells are readily identified by light microscopy and confirmed by a fluorescence-based method, giving an adhesion index that reveals the tumor cell-binding affinity to LN parenchyma.

Tumor-draining lymph nodes (LNs) are not merely filters of tumor-produced waste. They are one of the most common regional sites of provisional residence ...

Cancer Research

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal (central) efferent lymph. Importantly, the collection of central lymph from the intestine enables researchers to study both the mechanisms and lipid constitutes associated with lipid metabolism, intestinal inflammation and cancer metastasis, as well as cells involved in immune function and immunosurveillance. A ventral mid-line surgical approach permits excellent surgical exposure to the cranial abdomen and relatively easy access to the intestinal lymph trunk vessel that lies near the pancreas and the right ventral segment of the portal vein underneath the visceral aspect of the right liver lobe. The vessel is meticulously dissected and released from the surrounding fascia and then dilated with sutures allowing for insertion and subsequent securing of the catheter into the vessel. The catheter is exteriorized and approximately 1 L/24 hr of lymph is collected over a 7 day period. While this technique enables the collection of large quantities of central lymph over an extended period of time, the success depends on careful surgical dissection, tissue handling and close attention to proper surgical technique. This is particularly important with surgeries in young animals as the lymph vessels can easily tear, potentially leading to surgical and experimental failure. The video demonstrates an excellent surgical technique for the collection of intestinal lymph.

We present a surgical procedure to catheterize the intestinal lymph trunk in neonatal pigs to collect large quantities of lipid metabolism components from efferent lymph.

Catheterization of the intestinal lymph trunk in neonatal pigs is a technique allowing for the long-term collection of large quantities of intestinal ...

Medicine

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Laparoscopic Technique for Serial Collection of Liver and Mesenteric Lymph Nodes in Macaques

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them immunologically unique. The GI tract and associated MLN are sites of early viral replication in human immunodeficiency virus (HIV) infection and the MLN are likely important reservoir sites that harbor latently-infected cells even after prolonged antiretroviral therapy (ART). The liver has been shown to play a significant role in immune responses to lentiviruses and appears to play a significant role in clearance of virus from circulation. Nonhuman primate (NHP) models for HIV and Acquired Immunodeficiency Syndrome (AIDS) closely mimic these aspects of HIV infection and serial longitudinal sampling of primary sites of viral replication and the associated immune responses in this model will help to elucidate critical events in infection, pathogenesis, and the impact of various intervention strategies on these events. Current published techniques to sample liver and MLN together involve major surgery and/or necropsy, which limits the ability to investigate these important sites in a serial fashion in the same animal. We have previously described a laparoscopic technique for collection of MLN. Here, we describe a minimally invasive laparoscopic technique for serial longitudinal sampling of liver and MLN through the same two port locations required for the collection of MLN. The use of the same two ports minimizes the impact to the animals as no additional incisions are required. This technique can be used with increased sampling frequency compared to major abdominal surgery and reduces the potential for surgical complications and associated local and systemic inflammatory responses that could complicate interpretation of results. This procedure has potential to facilitate studies involving NHP models while improving animal welfare.

Here, we describe a minimally invasive laparoscopic technique for serial sampling of liver and mesenteric lymph nodes (MLN) in macaques that allows for increased sampling frequency, and reduces the potential for surgical complications when compared to performing a laparotomy.

The mesenteric lymph nodes (MLN) and the liver are exposed to microbes and microbial products from the gastrointestinal (GI) tract, making them ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Neuroscience

A Cryo-pulverization Protocol for Processing Mouse Paws to Evaluate Molecular Pathways of Tissue Inflammation in a Collagen Induced Arthritis Model

Profiling molecular changes in local tissues is crucial to understand the mechanism(s) of action of therapeutic candidates in vivo. In the field of arthritis research, many studies are focused on inflamed joints that are composed of a complex mixture of bone, cartilage, muscle, stromal cells and immune cells. Here, we established a reliable and robust mechanical method to disrupt inflamed mouse paws into homogeneous pulverized samples in a cryogenically controlled environment. Protein and RNA lysates were processed to enable proteomic and transcriptional endpoints and molecular characterization of relevant disease pathways in local tissue.

A cryogenic pulverization method to process murine paws using a liquid nitrogen freezer mill was developed to improve the yield and quality of RNA or protein extracted from the tissues and enable the analysis of molecular profiles associated with inflammatory responses.

Profiling molecular changes in local tissues is crucial to understand the mechanism(s) of action of therapeutic candidates in vivo. In the field of ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response against helminth infections, where it plays an important role in the expulsion of the parasite. In a murine model of Nippostrongylus brasiliensis infection, IL-9 is produced mainly by CD4+ T lymphocytes and innate lymphoid cells found in the lung, small intestine, and draining lymph nodes. Given the technical difficulties involved in the intracellular staining of IL-9, as well as the complexity of isolating hematopoietic cells from the small intestine upon infection, there is a pressing need for a comprehensive but straightforward protocol to analyze the expression of IL-9 in different lymphoid and non-lymphoid tissues in this model. The protocol described here outlines the kinetics of IL-9 produced by CD4+ T cells and innate lymphoid cells in the lung and small intestine, the main organs targeted by N. brasiliensis, as well as in the mediastinal and mesenteric lymph nodes, throughout the infection. In addition, it details the number of larvae needed for infection, depending on the cell type and organ of interest. This protocol aims to assist in the standardization of assays to save time and resources by offering the opportunity to focus on the specific cells, organs, and disease stages of interest in the N. brasiliensis infection model.

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9-expressing T and ILC2 cells are induced during N. brasiliensis infection, yet their characterization has been largely overlooked in the infected intestine due to their low frequency and differential kinetics. This protocol describes the isolation of these cells from different target organs and confirmation of their identity via flow cytometry at different infection stages.

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Summary

Explore More Videos

Chapters in this video

Mouse Adipose Tissue Collection and Processing for RNA Analysis

Quantification of Tumor Cell Adhesion in Lymph Node Cryosections

Isolation of Murine Lymph Node Stromal Cells

Long-Term Catheterization of the Intestinal Lymph Trunk and Collection of Lymph in Neonatal Pigs

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

Laparoscopic Technique for Serial Collection of Liver and Mesenteric Lymph Nodes in Macaques

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

A Cryo-pulverization Protocol for Processing Mouse Paws to Evaluate Molecular Pathways of Tissue Inflammation in a Collagen Induced Arthritis Model

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model