5 Sample preservation

Utilising liquid nitrogen for snap-freezing samples is widely regarded as the benchmark method for extracting pristine DNA from an unmodified microbial community. Nevertheless, the feasibility of promptly freezing samples while maintaining an unbroken cold chain isn’t always achievable, especially during field sampling missions. Consequently, dependable substitutes become imperative. This necessity has spurred the creation of various preservatives, enabling samples to be preserved at room temperature over prolonged durations. Nonetheless, research has unveiled that these preservatives can impart significant biases during the generation of diverse omic layers [15]. Therefore, maintaining uniformity in the selection of preservatives is of paramount importance to ensure consistency across analyses.

Preservative HG/MG HT/MT MP ME Reference
Snap frozen Yes Yes Yes Yes [16]
RNAlater Yes Yes Yes No [17]
DNA/RNA Shield Yes Yes No No [18]
OMNIgene GUT Yes Yes No No [19]
Tris-EDTA Buffer Yes Yes No No [20]
Guanidine thiocyanate Yes Yes No No [21]
TRIzol Yes Yes Yes No [22]
Protease inhibitors No No Yes No [23]
FTA Cards Yes Yes No Yes [24]
Methanol No No No Yes [25]
Ammonium bicarbonate No No Yes No [26]

Numerous preservatives necessitate removal before extraction, such as ethanol, NAP buffer, and RNAlater. However, this removal process can inadvertently eliminate non-pelleting entities, including bacteriophages and other viruses. Conversely, certain preservatives also serve as lysis buffers, exemplified by Zymo’s DNA/RNA Shield. Notably advantageous, these buffers directly participate in DNA extraction. Although these preservatives do not stabilise DNA within cells, they initiate cellular degradation while stabilising DNA in the matrix. Maintaining the recommended material-to-buffer ratio is imperative across all these buffers to ensure optimal outcomes. Overloading with biological material can negate the beneficial effects of the buffers.

Avoiding freeze-thaw cycles is ideal, as they are recognised sources of DNA degradation and variations in microbial community composition, especially when samples are repetitively thawed [27]. Opting for small aliquots tailored to the extraction protocol during sample collection, rather than bulk collection, facilitates thawing only the sample intended for processing. This practice sidesteps detrimental thaw-freeze cycles and diminishes the risk of cross-contamination from other samples.

Furthermore, the biological and chemical characteristics of molecules (e.g., DNA, RNA, proteins, metabolites) used in omic data generation must be acknowledged. Host DNA’s abundance and stability render HG less sensitive. Conversely, MG warrants more cautious handling due to potential microbial community fluctuations post-sampling, unless biochemical reactions are halted. HT and MT demand even swifter preservation to capture representative gene expression patterns. Lastly, metabolites exhibit diverse chemical properties, ranging from stable steroids to highly volatile short-chain fatty acids. Thus, judicious selection of appropriate preservatives becomes paramount when generating multiple omic layers. This decision involves determining whether omic data will be sourced from a single biological sample, necessitating a universal preservative, or multiple samples, each potentially requiring a distinct preservative.

Importantly, the diverse physicochemical properties of samples mandate that collection and storage methods validated for one sample type cannot be universally assumed optimal for others. Therefore, preliminary optimisation tests are prudent, and methodological consistency emerges as a prerequisite for the production of dependable omic data.

Contents of this section were created by Antton Alberdi, Ostaizka Aizpurua and Jacob A Rasmussen.


15. Pérez-Losada M, Crandall KA, Freishtat RJ. Two sampling methods yield distinct microbial signatures in the nasopharynges of asthmatic children. Microbiome. 2016;4:25.
16. De Spiegeleer M, De Graeve M, Huysman S, Vanderbeke A, Van Meulebroek L, Vanhaecke L. Impact of storage conditions on the human stool metabolome and lipidome: Preserving the most accurate fingerprint. Anal Chim Acta. 2020;1108:79–88.
17. Eijsden RGE van, Stassen C, Daenen L, Van Mulders SE, Bapat PM, Siewers V, et al. A universal fixation method based on quaternary ammonium salts (RNAlater) for omics-technologies: Saccharomyces cerevisiae as a case study. Biotechnol Lett. 2013;35:891–900.
18. Schweighardt AJ, Tate CM, Scott KA, Harper KA, Robertson JM. Evaluation of commercial kits for dual extraction of DNA and RNA from human body fluids. J Forensic Sci. 2015;60:157–65.
19. Wang Z, Zolnik CP, Qiu Y, Usyk M, Wang T, Strickler HD, et al. Comparison of fecal collection methods for microbiome and metabolomics studies. Front Cell Infect Microbiol. 2018;8:301.
20. Barra GB, Santa Rita TH, Almeida Vasques J de, Chianca CF, Nery LFA, Santana Soares Costa S. EDTA-mediated inhibition of DNases protects circulating cell-free DNA from ex vivo degradation in blood samples. Clin Biochem. 2015;48:976–81.
21. Weidner L, Laner-Plamberger S, Horner D, Pistorius C, Jurkin J, Karbiener M, et al. Sample buffer containing Guanidine-Hydrochloride combines biological safety and RNA preservation for SARS-CoV-2 molecular diagnostics. Diagnostics (Basel). 2022;12.
22. Simões AES, Pereira DM, Amaral JD, Nunes AF, Gomes SE, Rodrigues PM, et al. Efficient recovery of proteins from multiple source samples after trizol or trizolLS RNA extraction and long-term storage. BMC Genomics. 2013;14:1–15.
23. Ryan BJ, Henehan GT. Avoiding proteolysis during protein purification. Methods Mol Biol. 2017;1485:53–69.
24. Bolt Botnen A, Bjørnsen MB, Alberdi A, Gilbert MTP, Aizpurua O. A simplified protocol for DNA extraction from FTA cards for faecal microbiome studies. Heliyon. 2023;e12861.
25. Straughen JK, Sitarik AR, Jones AD, Li J, Allo G, Salafia C, et al. Comparison of methanol fixation versus cryopreservation of the placenta for metabolomics analysis. Sci Rep. 2023;13:4063.
26. Hedges JB, Vahidi S, Yue X, Konermann L. Effects of ammonium bicarbonate on the electrospray mass spectra of proteins: Evidence for bubble-induced unfolding. Anal Chem. 2013;85:6469–76.
27. Cuthbertson L, Rogers GB, Walker AW, Oliver A, Hoffman LR, Carroll MP, et al. Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses. J Cyst Fibros. 2015;14:464–7.