Location: Infectious Bacterial Diseases ResearchTitle: Collection and processing of lymph nodes from large animals for RNA analysis: preparing for lymph node transcriptomic studies of large animal species Author
|Schaut, Robert - US Department Of Agriculture (USDA)|
Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/2/2018
Publication Date: 5/19/2018
Citation: Vrentas, C.E., Boggiatto, P.M., Schaut, R.G., Olsen, S.C. 2018. Collection and processing of lymph nodes from large animals for RNA analysis: preparing for lymph node transcriptomic studies of large animal species. Journal of Visualized Experiments. 135:e57195. doi:10.3791/57195.
DOI: https://doi.org/10.3791/57195 Interpretive Summary: Improving our understanding of the ways in which hosts (like cattle and bison) respond to brucellosis infections is of significant interest for future work to design disease interventions, including new vaccines or therapeutics. One way to examine host responses is by characterizing changes in the pool of RNA molecules that are made from ("expressed from") DNA inside the cells of host lymph nodes. In order to do so, however, it is critical to isolate high-quality RNA from animal tissues. Experiments with large animal species, like livestock and wildlife, are necessarily more challenging to work with than laboratory animal models like mice or rats. Therefore, in this manuscript, we develop and then demonstrate protocols to aid researchers in (a) isolating lymph nodes from large animals like cattle, and (b) properly storing and isolating RNA from the tissue samples. We focus in particular on lymph nodes useful for brucellosis research, but the methodologies are applicable to a range of diseases. We also present new data that we have collected that examines the impact of delays (in the field or in the lab) before lymph node tissue collection on RNA quality, and examine the impact of preservation methods like formalin fixation/paraffin embedding on RNA integrity in lymph node sections.
Technical Abstract: Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Salmonella, and E. coli, as well as useful models for the study of pathogenesis and/or spread of the bacteria in non-murine hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of both host and pathogen RNA for downstream transcriptomic analysis, in order to assess temporal changes in gene expression in cells over the course of infection. This video protocol 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 American bison (Bison bison). The protocol includes information about location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for consistency of sampling across multiple animals. Considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the longer delays inherent in large animal vs. mouse time course studies, representative results from bison supramammary lymph node tissue are presented to describe the time course of 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 for both veterinary researchers beginning transcriptome projects on large animal samples and molecular biologists interested in learning techniques for veterinary tissue sampling and processing. Original research elements include data collected and presented relating to the time course of degradation of RNA in bovine and bison lymph node tissues, as well as the results from application of RNA extraction methodologies to alternatively preserved tissues.