Metabolomics of juvenile P. generosa - Sample preparation
Objective(s):
- Prepare geoduck metabolomic samples for analysis following K Wong’s test with larvae and adult coral samples link here.
- About our samples: Geoduck samples are whole individuals ~5-6 months in age snap frozen in LN2 and stored at -80C for ~1 year. Sizes vary ~5-8 mm shell length.
Important links/comments:
K.Wong’s notebook post on P. astreoides sample prep link here
MSDS links of harmful chemicals
- 40% Acetonitrile - fishersci MSDS link
- 40% Methanol - fishersci MSDS link
- less than 1% Formic Acid - fishersci MSDS link
- 15% Ammonium Bicarbonate - fishersci MSDS link
READ HERE!
- It is critical to take extreme caution with this protocol. Acetonitrile (or methyl cyanide) is very toxic causing harm due to skin/eye contact, inhalation, and ingestion. change gloves often if you notice any substance on them
- Even a subtle wet glove from the extraction buffer can lead to a burning skin iritation
- Read through MSDS and take proper precaution
Equipment needed:
PPE
- must be completed in a fume hood!
- lab coat
- gloves (long gloves prefered)
- glasses/goggles
Other equipment
- 2mL dounces Make sure dounces are autoclaved between each sample, alternatively use a new autoclaved dounce for each sample
- 11mm Snap Cap tubes
- 11mm Snap Cap auto sampler vials
- 100um cell strainer
- 1.5 ml tubes
- pippette tips
- 1 ml and 200 ul pipettes
- dry ice
Chemicals:
NOTE: the reagents below were already made by K.Wong and stored at 4°C in a non-flammable fridge; below is the method to create these stock solutions. same PPE requirments above applies to create more stock solution (i.e. fume hood, coat, gloves, goggles)
Reagent Preparation
(I) Extraction Buffer
This extraction buffer is a ratio of 40:40:20 of methanol, acetonitrile, and water, with 0.5%[v/v] Formic Acid
For a total of 200mL extraction buffer:
- 80mL methanol
- 80mL acetonitrile
- 40mL water
- 1mL formic acid
* Store at 4°C in a non-flammable fridge
(II) )15% Ammonium Bicarbonate Solution
Solubility of Ammonium bicarbonate in water is: 220g/L for 100%
100% Ammonium Bicarbonate solution (250mL)
- 55g dry Ammonium bicarbonate in 250mL LCMS water
15% Ammonium Bicarbonate Solution (250mL)
- 37.5mL of 100% Ammonium Bicarbonate Solution + 212.5mL LCMS water
* Store at 4°C in a non-flammable fridge
Preparation of space and samples before extraction
Prepare your fume hood and work space
To streamline your work it is ideal to set your fume hood and workspace with the following equpiment:
Inside the fume hood
- pipette tips and pipettes (1 ml, 200 ul)
- small box of dry ice
- 10% alkaline detergent in a 500 ml basin (i.e. tripour plastic beaker)
- 1 X n dounces
- 3 X n 1.5 ml tubes labelled with sample ID
- 2 X n autosampler vials with caps labled with sample ID
- 1 X n 100 um cell strainers
- 1 X n 50 ml disposable receptacle (i.e. 50 ml Falcon tube)
- alternatively 50 ml beakers can be used - if you do use glassware you will need a larger/multiple detergent basin(s)
- labeled toxic waste bag for all disposable equipment (pipette tips, Falcon tubes, cell strainers, 1.5 ml tubes, etc.)
- Chemicals! use small beaker (i.e. 50 ml) to store what you need
- Extraction buffer; 1500 ul * n
- 15% Ammonium bicarbonate; 44 ul * n
Outside the fume hood (access to the following)
- scale
- vortex
- mini centrifuge
- standard temperature-controlled centrifuge for 1.5 ml tubes
- labeled tube box stored at -80°C
Extraction Protocol
Juvenile geoduck (whole animal) sample prep
- Remove the sample(s) needed for a single extraction from the -80°C
- Label and weigh an empty 1.5 ml vial empty, then with 1mL of the extraction buffer
- Place the whole sample(s) into this vial
- Weigh the vial with the extraction buffer and goeduck sample(s).
- Cap the tube and place on dry ice 5 minutes.
Extraction
- Carefully transfer sample to the dounce
- Note: the dounce fits in a standard 1/5 ml tube rack. This can be used to free a hand and minimiize contact with the extraction buffer during transfer
- Homogenize the sample until the supernatant is visibly pigmented.
- Add an additional 500uL of cold extraction buffer to rinse the pestle. Plaste the pestle in the detergent basin
- Note: the beaker of extraction buffer can be placed intermittantly on the dry ice to cool, it does become frozen so be aware of how long your beaker has been on ice
- Strain the extract (total volume in dounce) through a 100um cell strainer into a 50mL receptacle (i.e. Falcon tube or glass beaker). Place the dounce into the detergent basin
- Transfer extract to a new labeled 1.5 mL tube. Dispose of or put aside 50 ml receptable (Falcon tube into waste bag; glass beaker into detergent basin)
- Vortex and centrifuge for 10 minutes at 15000g at 4°C.
- Remove 500uL of the supernatant to a new labeled 1.5mL tube.
- Add 44uL of 15% ammonium bicarbonate.
- Vortex and spin down.
- Add 100uL into duplicate, labeled autosampler vials.
- Store 1.5mL tube and autosampler vials at -80°C.
Final table
| Sample # | Geoduck ID | Number animals | Vial (g) | Vial + Extraction buffer (g) | Vial + Extraction buffer + geoduck sample(s) (g) | Vial labels |
|---|---|---|---|---|---|---|
| 1 | 684 | 1 | 1.02 | 1.9 | 2.14 | 1, 1-A, 1-B |
| 2 | 652 | 1 | 1.03 | 1.83 | 1.86 | 2, 2-A, 2-B |
| 3 | 621 | 1 | 1.04 | 1.88 | 1.95 | 3, 3-A, 3-B |
| 4 | 638,624 | 4 | 1.04 | 1.89 | 2.24 | 4, 4-A, 4-B |
| 5 | 612,632 | 4 | 1.03 | 1.89 | 2.38 | 5, 5-A, 5-B |
| 6 | 659,666 | 4 | 1.02 | 2.02 | 2.46 | 6, 6-A, 6-B |
NOTE: under ‘Vial labels’ the #-A #-B are autosampler vials (each with 100 ul) and the # is a 1.5 ml tube with the remaining sample