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

Summary

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.

Abstract

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.

Introduction

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 mammals^1,2. 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 zo....

Protocol

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

1. Before the necropsy, prepare the following materials for transport to the necropsy suite:
   1. 1.5 or 2 mL microcentrifuge tubes filled with ~ 1.0 mL of RNA preservation solution, in a rack or transport container. Be sure to follow the manufacturer's guidelines for the ratio of the volume of the preservation solution to the volume of the tissue.

Results

The use of the considerations presented in this article (steps 1 - 4 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 ratio provides a measure of the protein contamination, and the A₂₆₀/A₂₃₀ ratio provides another means of purity assessment that will detec.......

Discussion

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.......

Materials

List of materials used in this article

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