Sample to Insight

Gabriela Eckerland, Pieter Caesens, Marco PolidoriQIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany

Sample storage before genomic DNA extraction:A practical guide to preserving DNA integrity and purity

DNA integrity is critical for accurate and reliable results in genotyping assays. When samples are collected, transported, and stored inadequately, DNA integrity is compromised; affecting downstream analysis. Resampling dramatically increases the costs of a project and often the same sample is impossible to collect again.

Most biological fluids (e.g., plasma, serum, and urine) and stool samples can be stored at 2–8ºC for several hours, although freezing at –20 or –80ºC is recommended for long-term storage. Swabs can be stored dry at room temperature.

Formalin fixation 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, tissue removal and fixation should be done as quickly as possible.

QIAGEN, as part of PreAnalytiX®, has developed the PAXgene® Blood DNA System; an integrated blood collection

and DNA purification system (storage for up to 14 days at room temperature). For tissue samples, PAXgene Tissue Containers provide fixation and stabilization of human tissue specimens. Nucleic acids, proteins, and sample morphology can be stabilized for up to 7 days at room temperature.

Animal tissueFreshly harvested tissue can be immediately frozen and stored at –20 or –80ºC, or in liquid nitrogen. Animal and human tissues can also be fixed for storage in fixatives such as alcohol and formalin; however, long-term storage of tissues in formalin will result in chemical modification of the DNA. It is also possible to isolate DNA from paraffin-embedded tissue. Animal, yeast, and bacterial cell cultures Cells should be stored at –20 or –80ºC. Animal cell nuclei can be prepared and stored at –20ºC.

Plant tissueFresh leaves/needles from most plant species can be stored for up to 24 hours at 4ºC. Samples can be stored longer at –80ºC. All samples stored at 4ºC should be kept in a closed container to prevent dehydration.

If it is not practical to store frozen samples, plant tissue should be dried. To prevent DNA degradation, material should be completely desiccated in <24 hours. Dried samples should be kept in the dark at room temperature under desiccating or hermetic conditions for long-term storage. Disrupted plant material can be stored in a suitable lysis buffer for several months at ambient temperature. Fungal materialAfter preparation, harvested samples can be either directly frozen or freeze dried, and stored at –80ºC.

DNA is a relatively stable molecule. However, introduction of nucleases to DNA solutions should be avoided as these enzymes will degrade DNA. Genomic DNA consists of very large DNA molecules, which are fragile and can break easily. To ensure the integrity of genomic DNA, excessive and rough pipetting and vortexing should be avoided. DNA is subject to acid hydrolysis when stored in water, and should therefore be stored –20 or –70ºC under slightly basic conditions (e.g., Tris·Cl, pH 8.0 or TE buffer; see tables). Avoid repeated freeze-thawing as this will lead to precipitates.

Diluted solutions of nucleic acids (e.g., dilution series used as standards) should be stored in aliquots (in siliconized tubes, where possible) and thawed once only. This avoids adsorption of nucleic acids to the tube walls, which would reduce the concentration of nucleic acids in solution.

TE buffer

1 M Tris-Cl

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Trademarks: QIAGEN®, QIAamp®, QIAsymphony®, DNeasy® (QIAGEN Group); PAXgene®, PreAnalytiX® (PreAnalytiX GmbH). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law.

© 2015 QIAGEN, all rights reserved.

An anticoagulant should be added to blood samples for storage. For example, blood samples treated with heparin or EDTA can be stored at 2–8ºC for a few days or at –20 or –80ºC for a few weeks. Alternatively, blood samples can be treated with ACD Solution (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 2–8ºC or 1 month at –20ºC. For long-term storage, blood nuclei can be prepared and stored at –20ºC.

The PAXgene Blood DNA System is an integrated and standardized system for collection and stabilization of whole blood specimens and isolation of their genomic DNA. The system requires the combined use of PAXgene Blood DNA Tubes for blood collection and stabilization, followed by DNA isolation using the PAXgene Blood DNA Kit.

Blood is collected into PAXgene Blood DNA Tubes, which contain a proprietary blend of reagents that both prevents blood coagulation and stabilizes white blood cells. Blood samples can be stored in the tubes for up to 14 days at

room temperature. DNA is isolated from the tubes using appropriate buffers supplied in the PAXgene Blood DNA Kit. Highly pure genomic DNA is obtained, suitable for use in a wide range of downstream applications.

The PAXgene Tissue System consists of prefilled containers for fixation and stabilization of tissue samples. It offers simultaneous stabilization of molecular content and preservation of morphology. The system does not rely on formalin fixation, thus avoiding destructive cross-linking and degradation.

Using the PAXgene Tissue System, samples can be transported and stored for up to 7 days at room temperature, >4 weeks at 2–8°C, and for longer periods at –20 or –80ºC.

The traditional method of tissue fixation involves placing specimens in a formalin solution. The resulting chemical reaction leads to cross-links between biomolecules, including cross-links between nucleic acids, between proteins, and between nucleic acids and proteins. After fixation in formalin, tissue specimens are embedded in paraffin. 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.

Introduction

Storage tips by sample type Storage of purified genomic DNA Ordering information

Storage of blood samples Fixing and storage of tissue samples

Genotyping experiments. PAXgene Blood DNA Tubes.

Adjust to pH with HCl.

*Additional kit sizes and/or formats available; see www.qiagen.com.

The PAXgene Tissue System.

Component Amount per literv

Tris base 121.1 g

Component Amount per liter

1 M Tris·Cl, pH 7.4 10 ml

0.5 M EDTA, pH 8.0 2 ml

Product Contents Cat. No.

PAXgene Tissue Container (10) For collection, fixation, and stabilization of 10 samples: 10 Prefilled Reagent Containers, containing PAXgene Tissue Fix and PAXgene Tissue Stabilizer 765112

PAXgene Tissue FIX Container (10 x 50 ml)

For fixation of tissue specimen: 10 prefilled reagent Containers containing 50 ml of PAXgene Tissue FIX; to be used in conjunction with PAXgene Tissue STABILIZERv 765312

PAXgene Tissue STABILIZER (8 x 150 ml)

For stabilization of tissue specimens previously fixed in PAXgene Tissue FIX : 8 bottles of PAXgene Tissue STABILIZER concentrate, for 4 liters of PAXgene Tissue STABILIZER; to be used in conjunction with PAXgene Tissue FIX Container

765512

PAXgene Blood DNA Tubes (100) For blood collection prior to genomic DNA purification using the PAXgene Blood DNA Kit (100 tubes) 761125

PAXgene Blood DNA Kit (25) For purification of up to 500 µg genomic DNA from whole blood (25 preps) 761133

QIAamp® DNA Mini Kit (50)* For 50 DNA preps from genomic, mitochondrial, bacterial, parasite, or viral samples 51304

QIAamp DNA FFPE Tissue Kit (50) For purification of genomic FFPE tissues 56404

DNeasy® Blood & Tissue Kit (50) For purification of genomic DNA from animal and tissue samples 69504

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