Comparison of DNA preservation methods for environmental bacterial community samples

Abstract

Field collections of environmental samples, for example corals, for molecular microbial analyses present distinct challenges. The lack of laboratory facilities in remote locations is common, and preservation of microbial community DNA for later study is critical. A particular challenge is keeping samples frozen in transit. Five nucleic acid preservation methods that do not require cold storage were compared for effectiveness over time and ease of use. Mixed microbial communities of known composition were created and preserved by DNAgard(™), RNAlater(®), DMSO-EDTA-salt (DESS), FTA(®) cards, and FTA Elute(®) cards. Automated ribosomal intergenic spacer analysis and clone libraries were used to detect specific changes in the faux communities over weeks and months of storage. A previously known bias in FTA(®) cards that results in lower recovery of pure cultures of Gram-positive bacteria was also detected in mixed community samples. There appears to be a uniform bias across all five preservation methods against microorganisms with high G + C DNA. Overall, the liquid-based preservatives (DNAgard(™), RNAlater(®), and DESS) outperformed the card-based methods. No single liquid method clearly outperformed the others, leaving method choice to be based on experimental design, field facilities, shipping constraints, and allowable cost.

Distinct Bacterial Microbiomes Associate with the Deep-Sea Coral Eguchipsammia fistula from the Red Sea and from Aquaria Settings

“…Whether this is a consequence of functional restructuring of the coral holobiont or a parallel response of the associated bacterial communities to differences in environmental conditions remains to be determined. Of note, in situ coral samples were preserved in RNAlater, whereas ex situ corals were flash frozen in liquid nitrogen, and it has been shown that RNAlater can bias samples regarding recovery of some bacterial taxa (Gray et al, 2013;Salter et al, 2014). Accordingly, some of the differences we found may be due to the different preservation methods used.…”

Biopolymers for sample collection, protection, and preservation

“…DNAgard, another product of Biomatrica, was created to permeate the cells and preserve the DNA in a proprietary solution. This system does not require refrigeration or freezing to retain the stability of the DNA for long-term storage (Table 1) (Gray et al 2013).…”

“…Beside the step of cell poration, this technique also includes a step of freeze-drying, which is not readily available in field conditions. FTA technology (Whatman, GE Healthcare, Little Chalfont, UK) is based on the proprietary FTA cards that contain chemicals that lyse cells, denature proteins, and protect nucleic acids from damage (Table 1) (Gray et al 2013). A solution-based mix of high salts, RNAlater® (Ambion, Carlsbad, CA) (Table 1), preserves tissues for a long time depending on the temperature.…”

“…For extended storage, fresh samples are immersed in the solution, refrigerated overnight allowing saturation of the tissue, and frozen after that. RNAlater® has been demonstrated to (Gray et al 2013) provide greater DNA yield than FTA® cards. RNAlater® furthermore gives the efficient preservation of RNA (Gray et al 2013).…”

“…Most of the existing methods of cell preservation are focused on the nucleic acid preservation and not on the safeguarding of cells (Gray et al 2013). However, preservation of viable cells has definite advantages compared to the preservation of DNA, RNA only.…”