TITLE: DNA extraction from recently fertilised Atlantic salmon embryos for use in microsatellite validation of triploidy
AUTHORS: Callum Howard, John B. Taggart, Caroline R. Bradley, Alejandro P. Gutierrez, John F. Taylor, Paulo A. Prodöhl, Herve Migaud, Michaël Bekaert

[DESCRIPTION]
The current methods used for producing triploid Atlantic salmon are generally reliable but not infallible, and each batch of triploids must be validated to ensure consumer trust and licensing compliance. Microsatellites have recently been shown to offer a cheaper and more convenient alternative to traditional flow cytometry for triploidy validation in a commercial setting. However, incubating eggs to at least the eyed stage for microsatellite validation poses challenges, such as reduced quality and performance of triploids produced from later eggs in the stripping season. To address these issues, we propose another option: extracting DNA from recently fertilised eggs for use in conjunction with microsatellite validation. To achieve this, we have developed an optimized protocol for HotSHOT extraction that can rapidly and cheaply extract DNA from Atlantic salmon embryos, which can then be used for triploidy validation through microsatellites. Our approach offers a simpler and more cost-effective way to validate triploidy, without the need for skilled dissection or expensive kits.

[STEPS]
SECTION: DNA extraction
1. If eggs stored in ethanol, remove using forceps and place on clean tissue to remove excess ethanol.
SECTION: DNA extraction
2. Place embryos in a beaker of Tris-HCl (5 millimolar (mM), pH 8) for 15 min.
SECTION: DNA extraction
3. Remove the eggs and remove excess liquid with clean tissue.
SECTION: DNA extraction
4. For low throughput needs the eggs can then be placed into individual 1.5 mL screw cap tubes, for high throughput needs the eggs can be places, one per well, into a 2 mL deep 96-well plate.
SECTION: DNA extraction
5. Pierce the chorion by applying pressure using the end of the forceps.
 

SECTION: DNA extraction
6. Add 400 µL alkaline lysis buffer to each tube/well and seal.
SECTION: DNA extraction
7. Invert 5 times, and placed into either a heat block or a laboratory oven running at 90 °C for 30 min.
SECTION: DNA extraction
8. Remove and place on ice for 5 min.
SECTION: DNA extraction
9. Unseal and add an equal amount (400 µL) of neutralisation buffer.
SECTION: DNA extraction
10. Reseal and rapidly invert 10 times and then spin down briefly using a centrifuge.
SECTION: DNA extraction
11. Spin down for 14000 rpm, 30 s (or 20,000 g).
SECTION: DNA extraction
12. Collect the middle layer of the solution.
 

SECTION: DNA extraction
13. The DNA (middle layer) can now be used instantly, stored at 4 °C for up to a week, or stored at -18 °C for use later on.
SECTION: DNA quality and quantity assessments
14. 
 
Mix 0.5 µL of sample DNA (middle layer), 3 µL MyTaq HS mix (Bioline, USA), 0.6 picomolar (pM) of each primer (0.12 µL) and 2.26 µL ultrapure water in PCR tube or plate (10 µL total).
SECTION: DNA quality and quantity assessments
15. Perform PCR at the appropriate thermal cycle for gene of interest.

SECTION: DNA quality and quantity assessments
16. Load 2.5 µL of the PCR product into a 1.25% agarose gel with 5 µL of 1.5× loading dye (ThermoFisher Scientific, UK) in 0.5× TAE buffer.

SECTION: DNA quality and quantity assessments
15.1. In this case, 38 cycles of
95 °C for 15 s,
60 °C for 15 s and
72 °C for 40 s.
SECTION: DNA quality and quantity assessments
17. Migrate the gel with ethidium bromide and visualised under UV in a transilluminator for the quality of bands and the presence of smear or primer dimer.
SECTION: DNA quality and quantity assessments
18. 
 
Mix 1 µL of sample DNA (middle layer), 5 µL Sensifast SYBR No-ROX kit (Bioline, USA), 1 picomolar (pM) of each primer (0.2 µL), 3.6 µL ultrapure water in qPCR plate.

SECTION: DNA quality and quantity assessments
19. Perform qPCR starting by 95 °C for 3 min followed by the appropriate thermal cycle for gene of interest.
SECTION: DNA quality and quantity assessments
19.1. In this case, 40 cycles of
 95 °C for 15 s,
 60 °C for 15 s and
 72 °C for 30 s.
SECTION: Microsatellites validation assessment
20. 
 
Mix required quantity of PCR product with 30 µL of sample loading solution (SLS), and 0.35 µL of size standard (WellRED size standard, Eurofins, Germany) and add to well of capillary electrophoresis plate.
SECTION: Microsatellites validation assessment
21. Top each well off with one drop of mineral oil.
SECTION: Microsatellites validation assessment
22. Run capillary electrophoresis machine (Beckman Coulter CEQ 8000, Beckman Coulter, USA) according to the manufacturer’s instructions.