Best Practices for Shipping Samples and Libraries for Next Generation Sequencing
One of the most frequently asked questions by researchers ordering services on Genohub is what is the best storage and shipping solution to ship extracted nucleic acid sample or library. What you tend to hear from each lab manager can vary, so we decided to put together a unified standard. With over 100 service providers offering library preparation and sequencing services on Genohub shipping recommendations were diverse, but going through each one of these we’ve put together a set of best practices. If you’re ordering a sequencing service on Genohub or even from a provider outside of Genohub’s network, you can follow these guidelines and rest assured that your samples will have been prepared and shipped properly.
Shipping RNA Samples
Dry ice is by far the easiest and most recommended way to ship total RNA samples. If you can’t ship with dry ice an ethanol precipitate is also fine. If you have an absolutely pure total RNA sample you can probably ship at room temperature or leave the sample on your bench for 1 week. However the reality is that many preparations and isolations do not completely remove nucleases. If your sample is contaminated with nucleases, you’ll need to slow down the rate it chops your RNA by reducing temperature. Finally, remember that RNA can be fragmented in water with divalent cations and heat, so heating RNA in water is not a good idea either. See our recommendations for shipping RNA in dry ice (recommended) or an ethanol precipitate if you can’t ship with dry ice.
Dry Ice
For most RNA applications, you’ll need to ship total RNA. If you’re interested in small RNA species, don’t forget to use a method that preserves small RNAs. Clean the surface of all tubes and use nuclease-free tips. Use nuclease-free water for buffer preparation and reconstitution of your total RNA sample. See the Packing section below and follow instructions for shipping with dry ice.
Ethanol Precipitation
Re-suspend your total RNA in a 100 uL precipitate solution.
Add:
- 1/10th volume of 3M NaOAc, pH 5.2
- 3 volumes of 100% ethanol
Mix well by vortex. Follow packing instructions below, but instead of dry ice use wet ice packs to maintain a -20 to 4°C shipping temperature.
Shipping DNA Samples
Shipping DNA that has been dissolved or re-constituted in TE (10 mM Tris, pH 8, 0.1 mM EDTA) or 10 mM Tris pH 8.0 with 4°C wet ice packs (blue ice) is the recommended way to ship DNA samples to a service provider who will make your libraries. Some service providers worry about the EDTA in TE inhibiting enzymatic reactions during library prep. If you’re only using 0.1 mM EDTA, by the time your concentrated DNA is diluted, and then diluted again during the first step of library preparation, the concentration of EDTA is so low, there is no chance it’s going to have any effect on the enzymes in library preparation. If after this, you’re still worried, just reconstitute your DNA in 10 mM Tris pH 8.0 rather than TE.
Yes, you can probably get away with shipping a pure DNA sample in water at ambient temperature. You can also lyophilize your DNA or dry it down on paper, but make the life of your service provider easier and just ship on a wet ice pack. You can ship extracted DNA on dry ice, but there is no good reason for this.
Shipping Fresh Tissue
Fresh tissue should be snap frozen and shipped on dry ice. If you’d like a provider to extract RNA or DNA from this tissue or if you can’t ship on dry ice, a preservation solution is recommended, e.g. RNAlater™ or DNA/RNA Shield™. These are aqueous solutions composed of ammonium sulfate that co-precipitate RNA and cellular proteins, 1) rendering RNA physically inaccessible to nucleases, and 2) inhibiting nucleases. Ammonium sulfate precipitation is typically a method used for purifying proteins by altering their solubility. Protein solubility depends on the ionic strength of the solution. At low ionic strength, solubility of protein increases with increasing salt, at high ionic strength, protein is completely precipitated out of solution, also known as ‘salting-out’.
There are a few online protocols for making your own ammonium sulfate based precipitation solution. Here is an example protocol from Palumbi Lab at Stanford University.
Commercial or homemade RNA preservation will preserve RNA for up to 1 week at room temperature or 4 weeks at 4°C, allowing processing and shipping of samples without liquid nitrogen or dry ice.
Constructed Library
A constructed library is a very stable, double stranded PCR product. Most providers will want between 2 – 10 nM of that library for sequencing. If you’re shipping a library you’ve constructed yourself, reconstitute your library in ~10-50 µl of 10 mM Tris pH 8.0 buffer and use microcentrfuge tubes that are 'low-bind'. At very low concentrations, libraries tend to stick to tube walls.
Our recommendations for shipping constructed libraries are the same ones as shipping DNA samples.
Packing and Shipping
Place your DNA or RNA sample in a 1.5 or 2 mL screw cap microcentrifuge tube and seal with Parafilm. Pack the tube in a freezer box, 50 mL conical vial or some other method to protect it from breaking. Place into a thermo-stable shipping box. We recommend the Styrofoam be at least 1.5 inches thick. Fill the box with dry ice or wet ice. See RNA or DNA sections in this guide to determine whether dry ice or wet ice is recommended. Here are our rough recommendations for the quantity of ice to pack:
- Shipments within your continent: 4 kg
- Shipments between Europe and North America: 6 kg
- Shipments between Asia and North America 9 kg
- Shipments between South and North America 9 kg
In a thermostable box where the Styrofoam is at least 1.5 inches thick, 3-4 kg of dry ice should last for at least 48 hours.
Use the fastest available courier, e.g. FedEx, UPS, DHL or USPS. The carrier may ask that you fill out a commercial invoice. Label your contents as ‘non-hazardous research sample’. Label the box as ‘temperature sensitive, keep frozen’. Send the service provider a tracking number so they know when to expect your package.
For international shipping, attempt to ship on Monday or Tuesday to avoid delay associated with delivering on a weekend. Determine whether the country you are shipping to has a holiday. Shipping during a holiday can needlessly delay the time it takes for a service provider to unpack and properly store your samples.
Using these recommendations will completely ensure you’re properly shipping raw, extracted nucleic acid, tissue or libraries. If you’re looking to find and compare sequencing and library preparation quotes between service providers, use Genohub’s NGS search interface. Once you’ve narrowed down the quotation you’d like to move forward with, your project is confirmed and detailed shipping instructions and conditions are displayed.
Shipping to the United States
Shipments from around the world to the United States frequently arrive without any issue or hold up at customs. A small percentage of shipments do get held. To reduce this chance even further, include the following letter in your shipping documents both inside and outside the shipping container:
Contents of this package are non-Infectious, non-Hazardous, not an etilogic agent, not for human consumption. Shipment consists of sterile DNA for scientific analysis only. The material contains small pieces of genomic DNA suspended in sterile water. The material was not generated by microbial fermentation. The product is purified and does not contain animal or cell derived materials or additives. The material is non-toxic, non-hazardous and nonpathogenic. The end use is for research purposes only, namely the generation of sequencing data by:
[ENTER NAME, ADDRESS and PHONE NUMBER of SERVICE PROVIDER]and will be used for laboratory research purposes only. Material will be maintained within appropriate biological containment facilities, preventing exposure of material to plants animals or the public. This package is temperature sensitive and needs to be handled in a timely manner.
[ENTER your NAME AND CONTACT INFORMATION HERE]
The USDA may require an import permit for international tissue samples. DNA and RNA samples generally do not require this permit. To determine if your specific samples from your country require an import permit, please refer to the USDA's Animal Health Status of Regions page.