Name: collection and processing of lymph nodes from large animals for rna analysis: preparing for lymph node transcriptomic studies of large animal species
Uploaded: 2018-05-19T14:00:00
Description: 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.

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 animal necropsy procedures depicted here are covered under approved IACUC protocols at the National Animal Disease Center, Ames, IA. All experiments were conducted in accordance with the approved guidelines for animal care and welfare.
1. Pre-planning Before Necropsy
<ol>
	<li>Before the necropsy, prepare the following materials for transport to the necropsy suite:
	<ol>
		<li>1.5 or 2 mL microcentrifuge tubes filled with ~ 1.0 mL of RNA preservation solution, in a rack or transport cont...

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 border="0" cellpadding="0" cellspacing="0" style="width:1239px;" width="1240">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:787px;">RNA preservation solution (we used RNALater for all experiments)</td>
			<td style="width:167px;">ThermoFisher</td>
			<td style="width:119px;">AM7020</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 ml or 2 ml polypropylene microcentrifuge tubes&#160;</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable scalpels</td>
			<td style="width:167px;">Daigger Scientific</td>
			<td style="width:119px;">EF7281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue forceps, rat tooth</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">12-460-117</td>
			<td>Other tissue forceps available including curved tip, tapered edge, etc. , depends on user preference</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 mm punch biopsy needles</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">NC9949469</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sharps container (small and transportable for necropsy)</td>
			<td style="width:167px;">Stericycle</td>
			<td style="width:119px;">8900SA</td>
			<td>1 qt. size shown here</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cutting boards or disposable trays</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">09-002-24A</td>
			<td>Available in a variety of sizes, depends on user preference</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Personal protective equipment</td>
			<td style="width:167px;"></td>
			<td style="width:119px;"></td>
			<td>Varies with pathogen (gloves, respirator masks, goggles, etc.)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol-based RNA extraction reagent (we used TRIzol Reagent for all experiments)</td>
			<td style="width:167px;">ThermoFisher</td>
			<td style="width:119px;">15596026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silica column-based RNA extraction kit (we used the PureLink RNA Mini kit for all experiments)</td>
			<td style="width:167px;">ThermoFisher</td>
			<td style="width:119px;">12183018A</td>
			<td>Designed for up to 100 mg tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100% Ethanol (200 proof for molecular biology)</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">E7023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue homogenizer with enclosed homogenization tubes (we used the gentleMACS dissociator for all experiments)</td>
			<td style="width:167px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose (General, for gel electrophoresis)</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">A9539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:787px;">1X TBE</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">BP24301</td>
			<td>Can also make from scratch in the laboratory</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:787px;">Deionized formamide</td>
			<td style="width:167px;">EMD Millipore</td>
			<td style="width:119px;">S4117</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:787px;">Sodium dodecyl sulfate</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">L3771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:787px;">Bromophenol blue</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">114391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:787px;">Xylene cyanol&#160;</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">X4126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:787px;">EDTA (Ethylenediaminetetraacetic acid)</td>
			<td style="width:167px;">Sigma-Aldrich</td>
			<td style="width:119px;">EDS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:787px;">UV-Vis Spectrophotometer (we used the NanoDrop Spectrophotometer)</td>
			<td style="width:167px;">ThermoFisher</td>
			<td style="width:119px;">ND-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:787px;">Device for quantitative RNA assessment (we used the Bioanalyzer, with associated components and protocols)</td>
			<td style="width:167px;">Agilent</td>
			<td style="width:119px;">G2939BA</td>
			<td></td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:787px;">FFPE RNA extraction kit (we used the RecoverAll Total Nucleic Acid Isolation Kit for Formalin Fixed, Paraffin Embedded Tissue)</td>
			<td style="width:167px;">ThermoFisher</td>
			<td style="width:119px;">AM1975</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:787px;">Plastic spreader (L-shaped spreader)</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">14-665-231</td>
			<td>Only needed for sterility testing for samples from infected animals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:787px;">Necropsy knives</td>
			<td style="width:167px;">Livestock Concepts</td>
			<td style="width:119px;">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 ...

Research

JoVE Journal

Immunology and Infection

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

Phagocytic cells such as alveolar macrophages and lung dendritic cells (LDCs) continuously sample antigens from the alveolar spaces in the 
lungs. LDCs, ...

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

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 ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Intracellular Staining and Flow Cytometry to Identify Lymphocyte Subsets within Murine Aorta, Kidney and Lymph Nodes in a Model of Hypertension

It is now well known that T lymphocytes play a critical role in the development of several cardiovascular diseases1,2,3,4,5. For example, studies from our ...

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Automated Behavioral Analysis of Large C. elegans Populations Using a Wide Field-of-view Tracking Platform

Caenorhabditis elegans is a well-established animal model in biomedical research, widely employed in functional genomics and ageing studies. To assess the ...