DNA testing is an integral component of breeding- and animal registration programs. Collecting high-quality samples is the most important step in the DNA testing process. Insufficient or poor quality samples delay turnaround times, and increase costs associated with resampling, resubmission and retesting.
Given proper storage conditions, DNA is a robust and stable molecule that can be stored for several years.
By following the guidelines written below you can help ensure a successful DNA test result the first time.
BEFORE YOU START
- Confirm the type of sample required for the DNA test(s) you want to request. (Sample requirements may vary among laboratories depending on the technology it uses)
- Confirm the amount/volume of sample required for the DNA test(s) you want to request.
- Confirm the best storage- and shipment method for the sample type you are collecting.
- Request the relevant forms to accompany the samples to be submitted.
COMMON REASONS FOR INCORRECT / NO TEST RESULTS
- Data entry errors in the field
– Samples are mislabelled or numbers are transposed
✔ Verify that the animal ID is clearly labelled on each sample and that the
information on the samples correspond to the information on the request form.
- Biological contamination
– Cross contamination between animals or samples
– Foreign material, e.g. faecal matter, dirt, microbial growth (leads to DNA degradation and/or PCR inhibition)
✔ Hands should be cleaned between animals along with any sampling tools (e.g. pliers, needles, syringes, etc.) used
✔ Remove any foreign material before sampling (e.g. faecal matter in tail hair)
- Chemical contamination
– Dye/Pigment from animal tattoos
– Dipping agents
– Cleaning agents
✔ Avoid sampling an animal from a tattooed area
✔ Properly clean the area to be sampled beforehand
- Incorrect storage/shipping conditions
The most common reasons for DNA degradation are exposure to moisture (e.g. incorrect storage container), heat and sunlight (e.g. leaving samples in a vehicle in the sun), and physical cleavage through freeze-thaw cycles (improper frozen storage).
✔ Always confirm the best storage- and shipment method for the sample type you are collecting.
Insufficient sample volume
Always confirm the amount/volume of sample required for the DNA test(s) you want to request
SPECIFIC SAMPLE TYPES
Hair samples are the preferred sample type for DNA testing. They provide the safest and most economical option for DNA testing as they are non-invasive (not harmful to the animal when collected), easy to collect (no veterinarian required), easily stored at room temperature, and conveniently and affordably transported to the laboratory.
DNA is not contained in the hair shaft itself, but rather in the root bulb.
How to collect and ship hair samples
- Ensure the animal is not wet when collecting the sample. Moisture can promote mould and microbial growth, which may cause DNA in the sample to degrade or inhibit PCR procedures
- Remove any foreign matter (e.g. faeces or dirt) from the animal before sampling
- Take a sample of 30-40 hairs (pencil thickness) from a clean area (tail hair works really well). If sampling younger animals, a larger number of hairs should be collected as the roots are smaller and contain less DNA.
- Pull the hair out firmly against the direction of growth. Do not cut the hair or take a sample from broken or shed hair.
- Confirm the presence of roots on the collected hair.
- Place the collected hair sample in a clean, dry envelope. Do not place hair samples in a plastic bag as this may lead to moisture build-up.
- Store hair samples in a dry location at room temperature and out of direct sunlight until shipment to the laboratory.
- Samples can be shipped at room temperature.
- Insufficient amount of hair collected
- No roots on hair
- Not enough sample collected from animals under 6 months of age
- Foreign matter present in hair (e.g. faeces)
How to collect and ship blood samples
- Clean the area on the animal before sampling to avoid contamination from dirt and manure.
- Use a new needle for each animal. This eliminates cross-contamination. Rinsing needles is not enough.
- When collecting blood, each sample must be drawn into a collection tube containing EDTA (purple top). EDTA is an anticoagulant (prevents clotting) and preservative. Other anticoagulants, such as heparin (green top) and DMSO (red top), should be avoided.
- Refrigerate the blood sample as soon as possible between 4-10° C. Do not freeze the sample.
- Samples can be shipped with/without icepacks.
- Incorrect collection tube used (blood can clot or additive inhibits PCR procedures)
- Collection tube not inverted several times after blood sample was collected (inadequate mixing with the anticoagulant, blood clotted)
- Samples left in direct sunlight (can lead to DNA degradation)
Blood collection cards
A blood collection card is chemically treated paper that traps DNA and binds anything that would degrade the sample.
How to collect and ship blood collection cards
- Blood can be spotted from a needle prick or previously collected blood.
- When spotting collection cards from stored blood, invert the collection tube 4-5 times before spotting.
- For a needle prick, sample from locations with easy-to-find blood vessels such as the ear and the underside of the tail.
- Saturate the blood collection card with enough blood to fill the circle outlined on the card, but not so much that the card is soggy.
- Don’t let the cards touch once blood samples have been blotted, as this may cross-contaminate samples.
- Allow the blood collection card to air-dry before closing it. Do not dry the blood collection card in direct sunlight or by using a hair-dryer as it will damage the DNA.
- Samples can be shipped at room temperature.
- Insufficient blood spot (the circle should be filled and blood present on both sides of the card)
- Too much blood applied to the collection card (slow to dry, may lead to microbial growth, increased risk of contamination)
- Closing the blood collection card before the blood spot is dry (may lead to microbial growth)
How to collect and ship tissue samples
- Tissue samples can be collected through an ear notch or biopsy dart.
- Don’t punch through tattoos, as ink residue may interfere with results.
- Samples can be stored in an empty tube, a collection tube filled with ethanol, a dry envelope, or a sealable plastic bag (not ideal).
- If collected in a plastic bag, freeze the samples until shipment to the lab. Avoid freeze-thaw cycles as this will degrade the DNA. The samples should be shipped with ice packs to prevent microbial growth.
- Ear notches can be stored dry in a paper envelope.
- Insufficient sample size
- Sample(s) shipped in plastic bags without proper packaging (ice packs).
- Microbial growth in the samples as a result of moisture build-up
Semen straws are commonly used when testing AI sires, imported sires, old or deceased sires for which no other DNA sample is available.
An empty straw can also be used to extract DNA, however, a positive result cannot be guaranteed. The straws are flushed out with sterile saline and this allows for any residual semen to be eluted into a tube. (The stopper should be included in the sample)
How to ship semen samples
Before submitting semen samples, remove the straw from the liquid nitrogen (if applicable) and allow it to gently thaw in a refrigerator or at room temperature. (Leaving the sample at room temperature for long periods can cause mould to appear on the sample)
Use a proper shipping container to protect the straw against breakage. Package the semen sample either surrounded by cardboard or inserted into an inexpensive writing pen with the ink tube removed.
Samples can be shipped at room temperature, no liquid nitrogen is required.
Ship in a padded envelope or small box.
Semen straws damaged during shipment due to improper packaging