Protocol optimization for simultaneous DNA and RNA co-extraction from single hard tick specimens

Alessandra Cafiso; Giulia Chiappa; Camilla Luzzago; Anita Koni; Daniele Bonato; Xhelil Koleci; Chiara Bazzocchi

doi:10.1016/j.mex.2021.101315

Protocol optimization for simultaneous DNA and RNA co-extraction from single hard tick specimens

MethodsX. 2021 Mar 21:8:101315. doi: 10.1016/j.mex.2021.101315. eCollection 2021.

Authors

Alessandra Cafiso¹, Giulia Chiappa¹, Camilla Luzzago^{1

2}, Anita Koni³, Daniele Bonato¹, Xhelil Koleci³, Chiara Bazzocchi^{1

2

4}

Affiliations

¹ Department of Veterinary Medicine, Università degli Studi di Milano, Lodi, Italy.
² Coordinated Research Center "EpiSoMI", Università degli Studi di Milano, Milano, Italy.
³ Department of Veterinary Public Health, Faculty of Veterinary Medicine, Agricultural University of Tirana, Tirana, Albania.
⁴ Pediatric Clinical Research Center "Romeo ed Enrica Invernizzi", Università degli Studi di Milano, Milano, Italy.

Abstract

Hard ticks are important vectors of DNA- and RNA-based infectious microorganisms, but they also host complex microbial communities in which pathogens and symbionts can interact among each other and with the arthropod host itself. Molecular investigations on ticks and their hosted microorganisms are important for human and animal health. These analyses often imply the use of both DNA and RNA, with prompt preservation of nucleic acids after collection, and safe handling in case of low-level containment. Several commercial kits are available for the co-extraction of DNA and RNA; however, cost can be a limiting factor for the choice of this method, which also require additional reagents for nucleic acids preservation before extraction. Additionally, extraction buffers provided in commercial kits do not guarantee the safe handling in case of hazardous biological material. With the aim of epidemiological screenings for tick-borne pathogens and gene expression analyses focused on the relationship among ticks and their microbial communities, an optimized protocol for DNA and RNA co-extraction from single adult hard tick specimens (Ixodidae) has been developed using TRIzol^Ⓡ LS Reagent.A method for•DNA/RNA co-extraction from adult hard tick specimens;•Safe sample handling;•Obtaining DNA and RNA simultaneously for diagnostic procedures and RNA for gene expression/transcriptomic analyses.

Keywords: Co-extraction; Coinfection; DNA isolation; Nucleic acids; PCR; Purification; RNA isolation; Reverse transcription; Tick; Vector-borne pathogen.