Location: Livestock Arthropod Pests ResearchTitle: A method for the isolation of miRNAs from tick ex vivo salivary gland cultures and extracellular vesicles
|LEAL-GALVAN, BRENDA - Texas A&M University|
|HARVEY, CRISTINA - Texas A&M University|
|OLIVA CHAVEZ, ADELA - Texas A&M University|
Submitted to: The Journal of Visualized Experiments (JoVE)
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/23/2022
Publication Date: 4/6/2022
Citation: Leal-Galvan, B., Harvey, C., Thomas, D.B., Saelao, P., Oliva Chavez, A. 2022. A method for the isolation of miRNAs from tick ex vivo salivary gland cultures and extracellular vesicles. The Journal of Visualized Experiments (JoVE). https://doi.org/10.3791/63618.
Interpretive Summary: Ticks are ectoparasites that can vector multiple pathogens. Despite their importance to animal and human health, the molecular mechanisms and techniques available are lacking. This manuscript describes the development of a molecular technique to extract the smallest particles of tick RNA for next generation sequencing. microRNAs are short sequences of RNA that have been known to regulate gene expression and are in need of improved profiling. This paper demonstrates how to perform the technical expertise to sequence and analyze these microRNAs and how the data can be used for future research into tick biology.
Technical Abstract: Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of the ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve in inter- and intra-cellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures for the collection of tick saliva. The focus of this project is to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. Herein, we describe the materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands.