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.
Highlights
The current method is suitable for multiple molecular analyses, in particular epidemiological studies aimed at investigating the presence of DNA- and RNA-based tick-borne pathogens (TBPs) in the same sample
It is applicable to transcriptomics and gene expression analyses on ticks and their microbial communities, and supports studies aimed at understanding the tick metabolism and biological interactions between ticks and their symbionts, or between ticks and tick-borne pathogens, or both
Six interphase/organic phases were subjected to DNA purification using the commercial kit NucleoSpin R Tissue kit (Macherey-Nagel, Düren, Germany) to reduce the required procedure time and compare the quality of DNA samples isolated with both protocols
Summary
We describe a method for the co-extraction of DNA and RNA form single adult unengorged and semi-engorged tick specimens using TRIzol R LS Reagent (Invitrogen, Thermo Fisher Scientific Inc., Waltham, MA, USA; hereafter “TRIzol R LS”). Six interphase/organic phases (obtained respectively from the remaining six samples) were subjected to DNA purification using the commercial kit NucleoSpin R Tissue kit (Macherey-Nagel, Düren, Germany) to reduce the required procedure time (about 1.5 h versus 2.5 h circa needed in TRIzol R processing) and compare the quality of DNA samples isolated with both protocols.
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