Isolation of microRNAs from Tick <em>Ex Vivo</em> Salivary Gland Cultures and Extracellular Vesicles

Brenda Leal-Galvan; Cristina Harvey; Donald Thomas; Perot Saelao; Adela S. Oliva Chavez

doi:10.3791/63618

Aislamiento de microARN de cultivos de garrapatas ex vivo de glándulas salivales y vesíc...

JoVE Journal
Biology

A subscription to JoVE is required to view this content. Sign in or start your free trial.

JoVE Journal Biology

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

Aislamiento de microARN de cultivos de garrapatas ex vivo de glándulas salivales y vesículas extracelulares

Full Text

3,075 Views

08:03 min

April 6, 2022

DOI: 10.3791/63618-v

Brenda Leal-Galvan¹, Cristina Harvey¹, Donald Thomas², Perot Saelao³, Adela S. Oliva Chavez¹

¹Department of Entomology,Texas A&M University, ²USDA-ARS Cattle Fever Tick Research Laboratory, ³USDA-ARS Veterinary Pest Research Unit

Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This study presents a protocol for isolating microRNAs from tick salivary glands and purified extracellular vesicles, utilizing a minimal number of ticks. The method is adaptable across various tick species and life stages, allowing for quality microRNA extraction that is suitable for sequencing.

Key Study Components

Research Area

MicroRNA isolation
Tick physiology
Extracellular vesicle biology

Background

MicroRNAs are crucial for gene regulation and cellular communication.
Ticks serve as important models for studying host-parasite interactions.
Isolation techniques can influence the quality of extracted microRNAs.

Methods Used

Ultracentrifugation for extracellular vesicle purification
Ticks (multiple species and life stages)
MicroRNA extraction and cDNA library preparation

Main Results

Protocol allows the use of only 20 ticks, reducing costs and ethical concerns.
Successfully isolates microRNAs with minimal degradation for further analysis.
The method is validated for consistency across different tick genders and life stages.

Conclusions

This protocol provides a reliable and cost-effective approach for microRNA studies in ticks.
It enhances the ability to investigate microRNA functions in tick biology and host interactions.

Frequently Asked Questions

What are the advantages of this microRNA isolation method?

The method allows for the use of a reduced number of ticks, minimizing costs and ethical implications while yielding high-quality microRNAs.

Can this protocol be applied to different tick species?

Yes, the protocol is designed to be adaptable across multiple tick species and life stages.

What is the storage duration for isolated microRNAs?

Isolated microRNAs can be stored at -20 degrees Celsius for up to three years.

How does this method ensure the purity of microRNAs?

The protocol involves multiple rounds of centrifugation and filtration to remove contaminants and cellular debris.

What applications can the extracted microRNAs be used for?

Extracted microRNAs can be used for profiling and functional assays to study their roles in gene regulation and signalling pathways.

Are there any procedural steps to prevent contamination?

Yes, it is critical to use antibiotics and follow specific steps during preparation to avoid microbial contamination.

How is the quality of the isolated microRNAs assessed?

Quality is evaluated through analysis of small RNA bands, focusing on ribosomal RNA and microRNA presence.

El presente protocolo describe el aislamiento de microARN de las glándulas salivales de las garrapatas y las vesículas extracelulares purificadas. Este es un procedimiento universal que combina reactivos y suministros de uso común. El método también permite el uso de un pequeño número de garrapatas, lo que resulta en microARN de calidad que se pueden secuenciar fácilmente.

Este protocolo nos permite utilizar un número reducido de garrapatas, lo que reduce el costo de mantenimiento de la colonia y disminuye el número de vértebras necesarias para la alimentación de garrapatas. Este protocolo requiere aproximadamente 20 ticks. Sin embargo, la principal ventaja, aparte de usar un tamaño de muestra pequeño, es que este protocolo se puede aplicar a múltiples especies de garrapatas y diferentes etapas de la vida.

Aparte de las disecciones de garrapatas o la aplicación hacia las interacciones huésped-parásito, el efecto de la infección por patógenos en la secreción de microARN y el efecto de los cambios fisiológicos en el microARN secretado por las garrapatas. Para comenzar, prepare medios libres de vesículas agregando suero bovino fetal, caldo de fosfato de triptosa, concentrado de colesterol de lipoproteína al 10%, bicarbonato de sodio al 5%, HEPES y medio L15C300 en una botella centrífuga de policarbonato de 26.3 mililitros y ajuste el volumen según sea necesario. Ahora ultracentrifugar el medio a 100, 000 veces G a cuatro grados centígrados durante 18 horas.

View the full transcript and gain access to thousands of scientific videos