This protocol details the concurrent extraction of RNA and DNA using the Zymo Quick-DNA/RNA Miniprep Plus Kit on planulae (larvae) and newly settled individuals (spat) of the coral Stylophora pistillata.

Following tests of this kit by others e.g. Kevin Wong, Erin Chille, Federica Scucchia I worked with 20 planulae in each vial or 6 - 8 spat in each vial. 700 uL of RNA DNA Shield was added to each sample upon collection. Samples were stored at -20 degrees until processing.

Reagents preparation

1. Add 96 mL 100% ethanol (104 mL 95% ethanol) to the 24 mL DNA/RNA Wash Buffer concentrate before use. DNA/RNA Wash Buffer included with D7003T (Mini Prep Plus Kit) is supplied ready-to-use and does not require the addition of ethanol prior to use. Check kit contents and instructions to confirm prep steps.
2. Reconstitute the lyophilized (freeze-dried) DNase I as indicated on the vial prior to use. Mix by inversion. Store frozen aliquots.
3. Prepare the 10 mM Tris HCl following this protocol.

Sample Preparation

Adapted From E. Chille’s Protocol with the addition of bead beating:

1. To each sample tube, add 5 – 10 2 mm beads
2. Shake for 60secs horizontally in bead beater. After shaking, allow the sample liquid to settle for few minutes.
3. Remove 300 uL sample to new labelled tube. Keep rest of the sample as spare in the fridge.
4. To the 300 ul sample, add 30 µl of PK digestion buffer
5. Add 15 µl Proteinase K to each sample tube
6. Vortex tubes to mix and hold at room temperature for 30 mins
7. Add equal volume (345 µl) DNA/RNA lysis buffer to each sample tube
8. Finger flick to mix tubes
9. Centrifuge at 16,000 rcf (g) for 30 seconds

DNA Extraction

1. Set up yellow DNA spin columns and collection tubes, label appropriately
2. Warm elution liquids to 70 degrees °C (10mM Tris HCl pH. 8.0 and RNase free water)
3. Add 690 µl (total volume) of sample gently to the yellow DNA spin column
4. Centrifuge at 16,000 rcf (g) for 30 seconds
5. Important Save the flow through from this step: transfer to a new 1.5mL tube labelled for RNA. Move to the RNA protocol to compete DNase treatment (steps 1 – 14)
6. Add 400µl DNA/RNA Prep Buffer gently to the yellow DNA spin columns
7. Centrifuge at 16,000 rcf (g) for 30 seconds
8. Discard flow through (Zymo kit waste)
9. Add 700 µl DNA/RNA Wash Buffer gently to the yellow DNA spin columns
10. Centrifuge at 16,000 rcf (g) for 30 seconds
11. Discard flow through (Zymo kit waste)
12. Add 400 µl DNA/RNA Wash Buffer genetly to the yellow DNA spin columns
13. Centrifuge at 16,000 rcf (g) for 2 minutes
14. Discard flow through (Zymo kit waste)
15. Transfer yellow columns to new 1.5mL microcentrifuge tubes
16. Add 50 µl warmed 10 mM Tris HCl to each yellow DNA column by dripping slowly directly on the filer
17. Incubate at room temp for 5 minutes
18. Centrifuge at 16,000 rcf (g) for 30 seconds
19. Repeat steps 18-20 for a final elution volume of 100 µl. If concentrations are low, elute once in 50 uL total volume.
20. Label tubes, store at 4 °C if quantifying the same day or the next, if waiting longer store in -20 °C

RNA Extraction

Can do concurrently with DNA Extraction after DNA Extraction Step 7

1. Add equal volume (700 µl) 100% EtOH to the 1.5mL tubes labelled for RNA containing the original yellow column flow through
2. Vortex and spin down to mix
3. Add 700 µl of that liquid to the green RNA spin columns
4. Centrifuge at 16,000 rcf (g) for 30 seconds
5. Discard flow through (Zymo kit waste)
6. Add 700 µl to the green RNA spin columns (the rest from the 1.5mL RNA tubes)
7. Centrifuge at 16,000 rcf (g) for 30 seconds o Get DNase I from freezer
8. Discard flow through (Zymo kit waste)
9. Add 400 µl DNA/RNA Wash Buffer gently to each green RNA column
10. Centrifuge at 16,000 rcf (g) for 30 seconds
11. Discard flow through (Zymo kit waste)
12. Make DNase I treatment master mix: o 75µl DNA Digestion buffer x # of samples o 5µl DNase I x # of samples
13. Add 80 µl DNase I treatment master mix directly to the filter of the green RNA columns
14. Incubate at room temp for 15 minutes

From here: combine steps with the DNA samples

1. Add 400 µl DNA/RNA Prep Buffer gently to each column
2. Centrifuge at 16,000 rcf (g) for 30 seconds
3. Discard flow through (Zymo kit waste)
4. Add 700 µl DNA/RNA Wash Buffer gently to the green RNA spin columns
5. Centrifuge at 16,000 rcf (g) for 30 seconds
6. Discard flow through (Zymo kit waste)
7. Add 400 µl DNA/RNA Wash Buffer genetly to the green RNA spin columns
8. Centrifuge at 16,000 rcf (g) for 2 minutes
9. Discard flow through (Zymo kit waste)
10. Transfer green columns to new 1.5mL microcentrifuge tubes
11. Add 50µl warmed DNase/RNase free water to each green RNA column by dripping slowly directly on the filer
12. Incubate at room temp for 5 minutes
13. Centrifuge at 16,000 rcf (g) for 30 seconds
14. Repeat steps 25-27 for a final elution volume of 100µl. If concentrations are low, elute once in 50 uL total volume.
15. Aliquot 5 – 10 µl to save for Nanodrop and Tape Station to avoid freeze-thaw of your stock sample
16. Store all tubes in the -80 °C

Check the DNA and RNA quality on the NanoDrop and Tape Station

Experiment conducted in Eilat in 2021 as part of NSF BSF grant (Award Number: 1937770). This post details the RNA DNA quality testing following extractions from coral tissue with this protocol

Sample List:

Extraction #	Sample_ID	Collection_Date	Sample_Content	Sample_Quantity	Sample_conserv
1	Test1	13/06/2021	Adult	10 polyps	700 uL shield
2	Test2	13/06/2021	Adult	10 polyps	700 uL shield
3	SS_a	7/05/2021	Spat	6 - 8 ind.	700 uL shield
4	SS_b	7/05/2021	Spat	6 - 8 ind.	700 uL shield
5	SS_c	7/05/2021	Spat	6 - 8 ind.	700 uL shield
6	SD_a	7/05/2021	Spat	6 - 8 ind.	700 uL shield
7	SD_b	7/05/2021	Spat	6 - 8 ind.	700 uL shield
8	SD_c	7/05/2021	Spat	6 - 8 ind.	700 uL shield
9	ShP_a	7/05/2021	Planulae	20 planulae	700 uL shield
10	ShP_b	7/05/2021	Planulae	20 planulae	700 uL shield
11	ShP_c	7/05/2021	Planulae	20 planulae	700 uL shield
12	ShP_d	7/05/2021	Planulae	20 planulae	700 uL shield
13	ShP_e	7/05/2021	Planulae	20 planulae	700 uL shield
14	DeP_a	7/05/2021	Planulae	20 planulae	700 uL shield
15	DeP_b	7/05/2021	Planulae	20 planulae	700 uL shield
16	DeP_c	7/05/2021	Planulae	20 planulae	700 uL shield
17	DeP_d	7/05/2021	Planulae	20 planulae	700 uL shield
18	DeP_e	7/05/2021	Planulae	20 planulae	700 uL shield

Results - Quality Testing

After kit extraction of RNA and DNA, sample quality was tested on the NanoDrop (RNA and DNA) and TapeStation (RNA only).

Tape station data:

Eluted RNA and DNA were aliquoted to 2 x 20 uL and 1 x 10 uL and stored at -80 degrees

Using the EpoFix Resin and following the EMS instructions for the preparation of the resin.

Ten days old spats of the coral Stylophora pistillata were fixed with resin for synchrotron radiation imaging (BAMline) at BESSY II in Berlin.

Samples preparation

• Take one filtered tip (20 µl) and cryotube (2ml) per each sample. Cut the top part of the tip (this is where the spat will be placed) and the bottom part of the tip.
• Fit the bottom part of the tip in the lid of the cryotube. If necessary cut more of the bottom-tip to ease the interlock.
• Mix 15 parts of resin with 2 parts of hardener in a plastic/paper cup and stir. At this point bubbles will form, so heat the Epofix mixture to remove them.
• Put a drop of resin mix at the top of the tip using a Pasteur pipette.
• Gently place the coral spat at the top of the tip using a razor blade.
• Gently add a drop of resin mix on the coral spat, wait for the resin to drip down the pipette. Add a second drop.
• After 1 hour add a third drop, make sure that the resin doesn’t drip down (the resin is almost solidified at this point).

• After 24 hours insert the tip + cup into the cryotube and close it. Your samples are ready for synchrotron radiation imaging!

This list comprehends all biomineralziation-related genes known from literature. The first reference is the one related to the accession number/gene ID, then we put all other references where that specific gene/protein was previously detected/immunolocalized (chronological order).

List preview: