Handling DNA

Sample storage prior to extraction of genomic DNA

The quality of the starting material affects the quality and yield of the isolated DNA. The highest DNA yield and quality is achieved by purifying genomic DNA from freshly harvested tissues and cells. If samples cannot be processed immediately after harvesting, they should be stored under conditions that preserve DNA integrity. In general, genomic DNA yields will decrease if samples, particularly animal samples, are stored at either 2–8°C or –20°C without previous treatment. In addition, repeated freezing and thawing of frozen samples should be avoided as this will lead to genomic DNA of reduced size or to reduced yields of pathogen DNA (e.g., viral DNA). Recommendations for storage of different starting materials are discussed below.

An anticoagulant should be added to blood samples that will be stored. For example, blood samples treated with heparin or EDTA can be stored at 2–8°C for a few days or at –20°C or –80°C for a few weeks. Alternatively, blood samples can be treated with ACD Solution B (0.48% citric acid, 1.32% sodium citrate, 1.47% glucose; use 1 ml per 6 ml blood) and stored for at least 5 days at 2–8°C or 1 month at –20°C. For long-term storage, blood nuclei can be prepared and stored at –20°C.
Most biological fluids (e.g., plasma, serum, and urine) and stool samples can be stored at 2–8°C for several hours. Freezing at –20°C or –80°C is recommended for long-term storage. Swabs can be stored dry at room temperature.

Formalin fixation and paraffin embedding (FFPE) is another means of sample storage and is particularly relevant for clinical tissue samples. Depending on the tissue type, the speed at which biomolecules are degraded, induced, or modified following harvesting can vary. Therefore, the procedures for tissue removal and fixation should be done as quickly as possible.

Fixation of tissues involves placing specimens in a formalin solution, which can vary in composition (a typical 10% formalin solution may contain 3.7% formaldehyde as well as 1–1.5% methanol). The resulting chemical reaction leads to cross-links between biomolecules, including cross-links between nucleic acids, between proteins, and between nucleic acids and proteins. For optimal results, neutral-buffered formalin solution should be used instead of unbuffered or acidic formalin solutions. Neutral buffer slows down the degradation of formalin, whose degradation products are believed to contribute to impairing nucleic acid quality.

The ratio of formalin to tissue should be at least 10:1 to ensure optimal fixation. This is easy to achieve when working with small tissue specimens, such as needle biopsies. However, when dealing with large tissue samples there may be insufficient formalin for fixation. In this case, sections of the tissue should be cut for formalin fixation. Tissues should be fixed for no more than 24 hours to avoid overfixation.

After fixation in formalin, tissue specimens are embedded in paraffin, a process which consists of several steps. The first step is dehydration, where water is replaced by an alcohol, usually ethanol. This is followed by clearing, where the alcohol is replaced by xylene or a xylene substitute, and by impregnation, where xylene is replaced by paraffin. The final step is embedding, where the entire specimen is surrounded with paraffin. It is important that tissue specimens are fully dehydrated prior to impregnation, as residual water may lead to sample degradation. We recommend always using fresh alcohol and xylene, to avoid any possibility of carryover of water from previous uses. To ensure optimal recovery of usable DNA from FFPE samples, low-melting–temperature paraffin should be used instead. In addition, paraffin containing additives such as beeswax should be avoided, as they may interfere with recovery of biomolecules.
Freshly harvested tissue can be immediately frozen and stored at –20°C, –80°C, or in liquid nitrogen. Lysed tissue samples can be stored in a suitable lysis buffer for several months at ambient temperature.

Animal and human tissues can also be fixed for storage. We recommend using fixatives such as alcohol and formalin; however, long-term storage of tissues in formalin will result in chemical modification of the DNA. Fixatives that cause cross-linking, such as osmic acid, are not recommended if DNA will be isolated from the tissue. It is also possible to isolate DNA from paraffin-embedded tissue (see Other clinical samples).
Centrifuge harvested cell cultures, remove the supernatant, and then store the cells at –20°C or –80°C. Alternatively, animal cell nuclei can be prepared and stored at –20°C.
Fresh leaves and needles from most plant species can be stored for up to 24 hours at 4°C without affecting DNA quality or yield. In general, samples that will be stored for longer than 24 hours should be stored at –80°C. However, some samples (e.g., tree buds) can be stored for several days at 4°C. Tissues stored at 4°C should be kept in a closed container to prevent dehydration. Large samples (e.g., branches) can be stored in a plastic bag containing a wet paper towel. 

If it is not practical to store frozen samples, a number of methods are available for drying plant tissue, for example, silica gel, food dehydrators, or lyophilizers (3). To prevent DNA degradation, material should be completely desiccated in less than 24 hours. Dried samples should be kept in the dark at room temperature under desiccating or hermetic conditions for long-term storage. Depending on how the sample was handled, the DNA in herbarium and forensic samples may be degraded. Disrupted plant material can be stored in a suitable lysis buffer for several months at ambient temperature.
Mycelium should be harvested directly from a culture dish or liquid culture. For liquid cultures, the cells should be pelleted by centrifugation and the supernatant removed before DNA isolation or storage. Harvested samples can be either directly frozen or freeze dried, and stored at –80°C.
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