Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Summary

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

Abstract

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of 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 inter-and intracellular 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 to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

Introduction

Ticks are ectoparasites that vector many pathogens to wildlife, livestock, humans, and their pets^1,2. Tick feeding results in significant economic loss by causing damage to hide, reducing weight and milk production due to severe anemia, and the transmission of potentially deadly disease-causing pathogens^1,3,4,5. Current control practices for managing tick populations are focused on the use of acaricides. Nevertheless, the continuous emergence of acaricide resistance in ticks parasiti....

Protocol

All animal experiments were performed following animal usage protocol (AUP#2020-0026) approved by the institutional animal care and use committee (AICUC) at Texas A&M University. The tick species, Ixodes scapularis and Rhipicephalus (Boophilus) microplus, and New Zealand Male White Rabbits, 42-72 days of age, were used for the present study. I. scapularis was received from the Center for Disease Control (CDC) and Oklahoma State University, certified as pathogen-free. R. microp.......

Discussion

The current protocol provides a detailed methodology for extracting miRNA from salivary glands and EVs. However, there are important considerations, all of which are detailed in the notes for each section of this protocol. The capsule and mesh netting must be secured during tick feeding to prevent ticks from escaping. The capsule preparation and placement are described in Koga et al.⁴⁰. Several replicates of the tick dissections need to be done if an unsuitable sample is discarded. Additionally, s.......

Materials

Name	Company	Catalog Number	Comments
0.22 µm syringe filter	GenClone	25-240
1 µm nylon syringe filter	Tisch Scientific	283129028
1 inch black adhesive	Amazon	B00FQ937NM	Capsule
10 mL needeless syringe	Exelint	26265
3' and 5' Adapters	Illumina	20024906	NEXTFLEX Small RNA-Seq Kit
4 mm vannas scissors	Fine Science Tools	15000-08
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid	Sigma-Aldrich	1.1523
70Ti rotor	Beckman Coulter	337922
Amphotericin	Corning	30-003-CF
Beads	Illumina	20024906	NEXTFLEX Small RNA-Seq Kit
Bioanalyzer	Agilent	G2939BA
Bioanalyzer kit	Agilent	5067-1513
Centrifuge 5425	Eppendorf
Chloroform	Macron	UN1888
Cyverse Discovery Enviornment			https://cyverse.org/discovery-environment
Dissecting microscope	Nikon	SMZ745
Double-sideded carpet tape	amazon	‎286373
Falcon Tubes, 50 mL	VWR	21008-940
Fetal Bovine Serum	Gibco	FBS-02-0050
fine forceps	Excelta	5-S-SE
Foamies, 2 mm	Amazon	B004M5QGBQ	Capsule
Isoflurane	Phoenix Pharmaceuticals manfactured	193.33165.3
Ixodes scaplaris	CDC, Oklahoma State University
L15C300 medium	In-lab
lipoprotein-cholesterol concentrate	MPI	02191476-CF
Microscope slide	VWR	10118-596
miRDeep2			https://github.com/rajewsky-lab/mirdeep2
M-MuLV Reverse Transcriptase	Illumina	20024906	NEXTFLEX Small RNA-Seq Kit
molecular grade ethanol	Fischer Bioreagents	UN1170
multi-well 24 well tissue culture treated plate	Corning	353047
Nanopaticle Tracking Analyzer machine	Malvern Panalytical
Nanosep with 300K Omega filter	Pall Corporation	OD3003C33
NEXTFLEX Small RNA-Seq Kit v3	PerkinElmer
NextSeq 500/550 High Output Kit (75 cycles)	Illumina	20024906
Optima XPN 90 Ultracentrifuge	Beckman Coulter
Penicillin	Thermofischer Scientific	ICN19453780
Pippettes	Ependorff
polycarbonate centrifuge bottle	Beckman Coulter	355618
Qiagen miRNeasy kit	Qiagen	217084
QIAzol lysis reagent	Qiagen	79306
Qubit	Thermofisher	Q32880
Qubit kit	Thermofisher	Q10212
Rabbits	Charles River
Reverse Universal Primer	Illumina	20024906	NEXTFLEX Small RNA-Seq Kit
Rhipicephalus microplus	Cattle Fever Tick Research Labratoty
Rifampicin	Fischer Bioreagents	215544
RNAlater	Invitrogen	833280
RNAse free tubes	VWR	87003294
RNAse inhibitor	Thermo Fischer	11111729
RNAse/DNAse free water	Qiagen	217084
RNeasy Minelute spin column	Qiagen	217084	Qiagen miRNeasy kit
RPE Buffer	Qiagen	217084	Qiagen miRNeasy kit
RT Buffer	Illumina	20024906	NEXTFLEX Small RNA-seq kit
RT Forward Primer	Illumina	20024906	NEXTFLEX Small RNA-seq kit
RTE Buffer	Qiagen	217084	Qiagen miRNeasy kit
Sodium bicarbonate	Sigma-Aldrich	S6014-25G
Sorvall ST16	Thermo Fischer	75004380
Sterilized Gauze sponges	Covidien	2187
Sterilized PBS	Sigma	RNBK0694
streptomycin	thermofischer Scientific	15240062
TapeStation	Aligent	G2991BA
Tear Mender Instant Fabric and Leather Adhesive	Amazon	7.42836E+11	Capsule
Tissue Adhesive	3M VetBond
Triple Antibiotics	dechra	17033-122-75
Tryptose phosphate broth	BD	BD 260300