Optimized protocol for DNA/RNA co-extraction from adults of Dirofilaria immitis

Chiara Lucchetti,Marco Genchi,Luigi Venco,Chiara Bazzocchi,Laura H Kramer,Alice Vismarra

doi:10.1016/j.mex.2019.10.023

Chiara Lucchetti, Marco Genchi + Show 4 more

Open Access

https://doi.org/10.1016/j.mex.2019.10.023

Copy DOI

Abstract

Dirofilaria immitis, the etiologic agent of canine heartworm disease, like several other filarial nematodes, harbors the bacterial endosymbiont Wolbachia. To investigate metabolic and functional pathways of D. immitis and Wolbachia individually, along with their interactions, the use of both transcriptomic and genome analysis has becoming increasingly popular. Although several commercial kits are available for the single extraction of either DNA or RNA, no specific protocol has been described for simultaneous extraction of DNA and RNA from such a large organism like an adult D. immitis, where female worms generally reach ∼25 cm in length. More importantly, adult worms of D. immitis can only be obtained either through necropsy of experimentally infected dogs or by minimally-invasive surgical heartworm removal of naturally infected dogs. This makes each individual worm sample extremely important. Thus, in the context of a project aimed at the evaluation of both gene expression analysis and Wolbachia population assessment following different treatments, an optimized protocol for co-extraction of DNA and RNA from a single sample of adult D. immitis has been developed.•An optimized method for DNA/RNA co-extraction from large size nematodes using TRIzol® reagent.•Allows maximum exploitation of unique samples as adults of D. immitis.

Highlights

Following owners’ consent, 12 adult worms of D. immitis, respectively 6 females and 6 males, were collected during minimally-invasive surgical heartworm removal from two dogs previously diagnosed with patent D. immitis infection
A 500 mL of TRIzol1 Reagent was added to each tube and a second RNA extraction was performed following the manufacturer protocol
Co-extraction has never been described for nematodes and, to the authors’ knowledge, there are no reports of this procedure from a single adult nematode sample, with a large size such as that of D. immitis

Summary

Introduction

An optimized method for DNA/RNA co-extraction from large size nematodes using TRIzol1 reagent. Subject Area: More specific subject area: Method name: Name and reference of original method: Resource availability: Veterinary Science and Veterinary Medicine Veterinary Parasitology DNA/RNA co-extraction from a single adult worm of D. immitis TRIzol1 Reagent protocol. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples.

Objectives

Results

Conclusion