Protocols for eDNA/eRNA extraction from marine samples
Luca Mirimin, Dulaney Miller, Sara Fernandez
Abstract
This document provides a series of protocols used to extract eDNA or eRNA from marine environmental samples such as small and large volume (filtered) water, sediment or (fine mesh net) plankton. Specifically, these protocols are recommended for use following sampling as described in Mirmin et al. (2021; Environmental DNA sampling protocols for the surveillance of marine non-indigenous species. protocols.io https://dx.doi.org/10.17504/protocols.io.by7rpzm6).
Before start
To minimize risk of sample contamination, note that all protocols should be carried out in dedicated eDNA extraction laboratories/rooms.
Ensure that each protocol and list of materials is checked before starting any of the procedures. Specifically, make sure that all key materials(e.g. kits) have not been modified or discontinued by the relevant supplier.
Steps
INTRODUCTION
Note that these nucleic acid extraction protocols have been adapted and tested in conjunction with sampling protocols as detailed in:
Environmental DNA sampling protocols for the surveillance of marine non-indigenous species
See also the relevant peer-reviewed publication here:
Overview of protocols included in this document:
| A | B |
|---|---|
| Protocol title | Description/purpose |
| PROTOCOL 1 - eDNA extraction from water samples (low volume water) | Extracting eDNA from filter membranes following filtration of low voume marine water (e.g. 1L) |
| PROTOCOL 2 - eDNA extraction from water samples (high volume water) | Extracting eDNA from High Volume filter capsules (e.g. 1 μm polyethersulfone filter membrane with an Effective Filtration Area of 1,300 cm2) |
| PROTOCOL 3 - eDNA/eRNA co-extraction from water samples (high volume water) | Co-extracting eDNA and eRNA from filter membranes following filtration of marine water |
| PROTOCOL 4 - eDNA extraction from water samples (high volume tow net) | Extracting eDNA from marine samples collected with a fine mesh (e.g. 50um) plankton net |
| PROTOCOL 5 - eDNA extraction from sediment samples | Extracting eDNA from marine sediment |
PROTOCOL 1 - eDNA extraction from water samples (low volume water)
Extract eDNA using the
Following sample collection as per " Protocol for collection of water samples (low volume water) ", cut each filter membrane in half with scissors and place it in a 10mL tube containing 0.25 g of 0.1 mm glass beads and 0.25 g of 0.5 mm glass beads
Add 720µL of 950µL of distilled water and 100 μL of Proteinase K
Bead beat the mixture for 0h 15m 0s at half speed using a
Equipment
| Value | Label |
|---|---|
| Mixer Mill MM 400 | NAME |
| Retsch | BRAND |
| MM400 | SKU |
(or equivalent)
Follow subsequent steps as per manufacturer's recommendations
During the final elution phase, add 100µL of
Repeat the step above for a final total volume of 200µL.
Store extract at -20°C .
PROTOCOL 2 - eDNA extraction from water samples (high volume water)
Extract eDNA using the
PROTOCOL 3 - eDNA/eRNA co-extraction from water samples (high volume water)
Co-extract eDNA and eRNA using
Store eDNA at -20°C and eRNA at -80°C .
Reverse Transcribe eRNA into cDNA using
cDNA can be stored at -20°C .
PROTOCOL 4 - eDNA extraction from water samples (high volume tow net)
Extract eDNA using the
Following sample collection as per " Protocol for collection of water samples (high volume tow net) ", mix by briefly vortexing and place a 15mL sub-sample into a new clean 50mL falcon tube (conical bottom). Centrifuge at max speed to form a pellet. Remove supernatant by pipetting.
Add 1350µL of 100µL
During the final elution phase, add 100µL of
Repeat the step above for a final total volume of 200µL.
Store extract at -20°C .
PROTOCOL 5 - eDNA extraction from sediment samples
Extract eDNA from a 5g sub-sample using
