TITLE: Total lipid content analysis for coral samples
AUTHORS: Keyla Plichon, Paola Furla

[DESCRIPTION]
This protocol aims to extract and quantify total lipidic content from frozen coral samples. This method is adapted from the extraction protocol of Rowan et al., 2020 and the sulfo-phospho-vanillin protocol on 96-well plate published by Cheng et al., 2011. Lipid content is normalized by the animal tissue surface determined by the aluminum technique described by Hoegh-Guldberg, 1988. The method was developed with the guidance of Dr. Mohamed Mehiri and Dr. Louis-Félix Nothias at Université Côte d'Azur.




[BEFORE_START]
During extraction, all the material used should be made of glass. All glassware should be washed and rinsed in 100% chloroform and allowed to dry in the fume hood before use. 

[GUIDELINES]
The protocol takes at least three days to be achieved, depending on the amount of samples.

[STEPS]
SECTION: Lipid extraction
3. Remove subsamples from the freeze dryer.
Turn off the freeze-dryer and quickly tighten the screw caps to the vial to avoid moisture entering the subsample. Keep samples at -80°C until analysis.
SECTION: Lipid quantifcation sulfo-phospho-vanillin method
12. Prepare a stock of cholesterol solution and prepare a standard range.
Prepare a stock of a 2 mg/ml cholesterol solution diluted in the extraction solvent. Dilute the stock solution to obtain a range of standards of 0, 0.125, 0.25, 0.5, 1, 1.25 & 1.5 mg/mL. The cholesterol solution should be prepared daily.
SECTION: Sample preparation
1. Weigh and rinse the coral subsamples. 
Make sure to keep the sample in liquid nitrogen, and cut a subsample of 1 cm2 with the help of a mortar and a pestle, making sure not to grind it. Rinse the subsample for 5 minutes in MilliQ water to remove salts. Place the subsample in the freeze-drying vial. Return the vial to the freezer for later.  
SECTION: Lipid extraction
4. Prepare solvents
Three solvents are needed throughout the procedure. On each extraction day, prepare:
the 2:1  (v/v) chloroform:methanol + 50 mg/L butylated hydroxytoluene (BHT) extraction solvent

 

a stock container with 100% chloroform and a stock container with 100% methanol

 

These solvents will be used for lipid extraction and rinsing.

SECTION: Lipid extraction
5. Prepare a potassium chloride, 0.88% solution (daily). 
Dissolve the KCl powder in Milli-Q water. This solution will be used for purification.


 

SECTION: Lipid extraction
6. Extract lipids.
Pour 10 mL of the chloroform:methanol solution into the glass extraction vial containing the freeze-dried sample. Record the time of solvent addition. Sonicate five times for 10s. Keep the vial on ice during sonication and make pauses between if needed, as it will cause heat. Store the vial in the dark for one hour at room temperature. 
SECTION: Lipid extraction
7. Filter subsamples.
After one hour, pour the supernatant containing the lipid extract from the extraction vial into a glass centrifuge tube. Using a glass syringe, dispense 2 mL of the extraction solvent into the extraction vial to collect the remaining lipids and pour the rinse into the centrifuge tube. Centrifuge the sample and pour the lipid extract into a clean glass vial or a separatory funnel. 

3000 rpm, 5 min 

SECTION: Lipid extraction
8. Purify lipid extracts.
Pour the lipid extract into a separatory funnel, the stopcock in the closed position. Add 4 ml of the 0.88 % KCl solution and place the stopper. Gently invert the funnel three times, making sure to release pressure. 
SECTION: Lipid extraction
9. Drain lipid extract.
Remove the stopper and drain the lower organic phase into a pre-weight clean vial, making sure not to let pass the aqueous (white) phase. Rinse the remaining aqueous with 1 mL of 100% chloroform and repeat the purification step. 
SECTION: Lipid extraction
10. Add methanol.
Add 1 mL of 100% methanol to the organic phase, the extract should now be transparent, otherwise, an additional 1 mL of methanol can be added. 
SECTION: Lipid extraction
11. Evaporate solvent.
Leave the vial containing the lipid extracts under the hood to let evaporate the solvent overnight between 37 °C and 40 °C. Do not overheat the extract, as heat may alter the lipids.

 

SECTION: Lipid quantifcation sulfo-phospho-vanillin method
13. Prepare a stock of 17% phosphoric acid.
Dilute the concentrated (100% or 80%)  phosphoric acid solution in Milli-Q water.
SECTION: Lipid quantifcation sulfo-phospho-vanillin method
14. Prepare a stock of 0.2 mg/mL vanillin reagent.
Dilute the vanillin in the 17% phosphoric acid solution. This solution corresponds to the reagent solution.

 

SECTION: Lipid quantifcation sulfo-phospho-vanillin method
15. Prepare a stock of concentrated (100%) sulfuric acid.
Keep a small amount of sulfuric acid at room temperature.

 

SECTION: Lipid quantifcation sulfo-phospho-vanillin method
16. Resuspend lipid extract.
Add 4 mL of the extraction solvent to the dried lipids' vial. 
SECTION: Lipid quantifcation sulfo-phospho-vanillin method
17. Prepare samples. 
Pour 150 μL of standard or sample into clean glass tubes (two per sample for duplicates and to speed up the process), and let evaporate the solvent in a water bath at 90°C for 30 min. 
SECTION: Lipid quantifcation sulfo-phospho-vanillin method
18. Add the sulfuric acid.
After completely evaporating the solvent, cool the tubes on ice for 5 minutes. Add 150 μL of sulfuric acid and place the samples back in a water bath at 90 °C for 20 min.
SECTION: Lipid quantifcation sulfo-phospho-vanillin method
19. Cool the tubes.
Place the tubes in an ice bath for 2 min.
SECTION: Lipid quantifcation sulfo-phospho-vanillin method
22. Add the reagent.
Add 50 μL of vanillin-phosphoric acid solution into each well.
SECTION: Lipid quantifcation sulfo-phospho-vanillin method
23. Incubate samples.
Incubate at 37°C for 15 min.

 

SECTION: Lipid quantifcation sulfo-phospho-vanillin method
24. Measure the absorbance at 540 nm.
Mix the samples just before reading absorbance.
SECTION: Lipid quantifcation sulfo-phospho-vanillin method
25. Determine lipid content.
Subtract the background and the blank from each sample and standard value. Compute the standard curve and calculate the lipid concentration for each sample. Multiply by the total volume. Some samples must be diluted to ensure they are within the standard range. 
  
 
determining lipid concentration
2. Dehydrate the subsamples.
In the late afternoon, turn on the freeze dryer, load subsamples into it, start the vacuum, and let them dry overnight. The protocol can be stopped here if necessary.
SECTION: Lipid quantifcation sulfo-phospho-vanillin method
20. Transfer the samples into a 96-well plate.
Transfer 100 μL of each sample. 

 
SECTION: Lipid quantifcation sulfo-phospho-vanillin method
21. Read the background absorbance in the spectrophotometer.
Determine the background at 540 nm.
SECTION: Normalization
26. Prepare aluminum range.
Cut pieces of aluminum with different known surfaces and weigh them with a balance accurate to 4 decimal places to create a standard curve.

 
The remaining samples can be kept at -80°C for further analyses.
SECTION: Normalization
27. Determine animal tissue coverage.
Cut pieces of aluminum and cover the skeleton surface that was covered with the animal tissue. Weigh the aluminum pieces and determine the corresponding surface with the standard curve. 
SECTION: Normalization
28. Normalize by surface.
Divide the total lipid content by the surface of the sample.