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Biosamples, from which DNA and RNA are extracted, are the essence of genomic and translational research. However, preservation of this precious research ingredient is often underestimated or poorly thought out. Furthermore, not many options other than cryogenic storage have been available for collection, storage and transport of biosamples as well as for multisite collaboration using these biosamples.

GenPlates are a proprietary format for storing and transporting biosamples at room temperature, in the dry state. GenPlates are high-density, 384-well plates that contain 6-mm discs of FTA™ paper (Whatman) molded into a hemispherical shape (Fig. 1). FTA is a macroporous cellulose filter paper that is chemically treated to inactivate mold, bacteria and viruses. Upon application of up to 10 μl of DNA or blood directly to the 6-mm FTA paper hemisphere, cells lyse, cellular enzymes are inactivated, and DNA is released to become entwined in the fibrous cellulose pores. The GenPlate that contains the entwined biosample is air-dried at room temperature and sealed with an adhesive cover and is then ready for storage or transport.

Figure 1
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A six-region GenPlate for storing DNA, blood and bacterial clones at room temperature in the dry state.

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When DNA is required for downstream analysis, high-quality double-stranded DNA from FTA paper or from neonatal-screening (Guthrie) cards is reliably recovered using GenSolve, a proprietary chemistry formulated by GenVault that allows safe recovery of high-molecular-weight, double-stranded DNA from GenPlates in less than 1.5 hours and in four easy steps (Figs. 2 and 3). GenSolve also facilitates the recovery of intact DNA from blood samples stored on Guthrie cards and FTA paper cards, thus enabling the use of Guthrie cards as a reliable source of DNA for genetic testing and population genetics, including methods such as short tandem repeat (STR) analysis, human leukocyte antigen (HLA) typing and genotyping assays from all common analysis platforms, including Illumina and Affymetrix.

Figure 2: High-quality genomic DNA.
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DNA from ten unique neonatal blood spots stored on Guthrie cards was recovered using GenSolve. Fragment length and integrity was assessed by running 30 ng of unamplified DNA from each sample directly on an agarose gel.

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Figure 3: PCR amplification.
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Endpoint amplification analysis of a 558-bp amplicon. Each lane shows a unique sample; the last lane is a negative control.

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GenTegra DNA Tubes is another proprietary technology for storing purified DNA in a 'bone-dry', water-free environment. This new matrix protects DNA samples from hydrolysis and oxidation with the added benefit of being simple and easy to use in a day-to-day laboratory environment. Just add purified DNA to the GenTegra DNA Tubes, dry and store. When needed, simply rehydrate, and the DNA sample is ready for downstream analysis. Nearly 99% of the originally stored DNA is recovered with the highest integrity.

GenConnect biosample inventory management software is a custom-configurable system that can be used to organize, track and mine data on biosamples stored in any type of container, at any temperature and in any format. This relational database software package is built on an open-source database, is easily scalable, can interface with existing applications by means of .csv and .txt file formats, and can be integrated with larger laboratory information management systems, thus allowing dry-state and frozen-state biobanking to be performed in parallel by a very large user group.

Bone-dry storage for preserving DNA and RNA integrity

Contrary to popular intuition, it can be argued that nucleic acids such as DNA and RNA are best preserved in an air-dried, water-free environment. DNA is a chemical polymer that is unstable in the presence of water because of its sensitivity to the addition of water across its phosphodiester bonds, which produces strand breakage1. RNA is also susceptible to strand breakage via hydrolysis in alkaline solutions. If water is absent, the rate of strand breakage becomes very slow for both RNA and DNA. Furthermore, extensive hydration is a severe limitation for long-term storage in an environmental sense because yeast, mold and bacteria can grow quickly in fluid media and water-saturated solids2. Thus, even embedding the nucleic acid in a polymer hydrogel or agar is not optimal for long-term storage. Eventually, bacteria and mold will grow on such solid gels because such matrices are, in general, well-hydrated when in equilibrium with ordinary relative humidity. As a result of these two summed effects (hydrolysis and microbial growth), if nucleic acids are to be stored in a moist environment, they must be refrigerated.

However, when air-dried in a matrix with low water content, both DNA and RNA become much more stable, in a chemical sense, at room temperature. Also, in a structural sense, because DNA and RNA are simple chemical polymers, water is generally unnecessary for retention of secondary structure, especially for the simple RNAs, where folding is not of functional importance. Thus, air drying has very little effect on the function of RNA or DNA that had been stored at room temperature.

The absence of water also leads to a second favorable consequence: the removal of water diminishes the rate of UV-light damage, as first noticed in the unusual stability of DNA in air-dried seeds3. In the air-dried state at less than 70% relative humidity, DNA remains a double helix but shifts to the A form of the helix. The A-form DNA, from top down, looks like a corkscrew, with the base pairs in a coiled configuration that is incompatible with ordinary UV light–induced thymidine dimer formation4. In the absence of that primary lesion, the only remaining source of UV-light photodamage in DNA is pyrimidine photohydrate formation, which is also slowed in the absence of water to react with the pyrimidine bases. At the heart of it, water can be seen as the main reason that, traditionally, DNA (or RNA) must be stored with refrigeration. In the absence of water, much of the rationale for cryogenic storage is eliminated.

Conclusion

GenVault's innovations in biosample management are providing researchers, lab managers and project directors with proven and tested alternatives to low-temperature storage. These innovations are resulting in streamlined costs, more efficient transport and storage, and a lower carbon footprint for a more sustainable and environmentally conscious research effort. More information about GenVault products is available at http://www.genvault.com/.