This post details RNA extractions for the 2023 Moorea Symbiotic Exchange project, which contains both larval and recruit samples.

RNA Extraction Protocol

The protocol for this work is documented in my notebook here.

The sample metadata is on GitHub here.

20250613

Today I transferred tissue samples for all larval samples (“A” and “P” sample IDs) from original screw cap cryovials into 1.5 mL safe-lock RNA free Eppendorf tubes.

• Thaw samples on ice (stored in RNA/DNA shield)
• Transfer to new tube labeled with sequencing ID
• Add new RNA/DNA shield to bring total volume to 1000 µL. Use the new RNA/DNA shield to ensure all material is gathered from the original tube.
• Stored at -80°C.

I’ll next complete this process for the remaining recruit samples.

20250708

Today I finished transfering all samples to the 1.5 mL labeled safe-lock tubes as done on 20250613. I transferred any tissue or skeletal samples along with the liquid for each tube.

I then froze all samples at -80°C.

Next I processed the 10 test samples (T1-T10) through bead beating and RNA extraction. I used the protocol described in my notebook here. For these test samples, I only processed RNA and did not proceed with DNA extraction.

The bead beating and extraction process was as described in the protocol. I ran the bead beater at the 8 setting (medium-high) for 2 minutes and then spun down all samples at 2000g for 1 min in a centrifuge. 500 µL were then transferred to a new 1.5 mL tube for RNA extraction. Extra material and beads were frozen as well for back ups.

After extraction (took about 2.5 hrs to do 10 samples), I quantified concentration using the Qubit.

Here are the results:

T1		ACR-A6	1	Acropora	Adult	800 ul shield	T1 RNA	20250708	80	58.6	4688	rna extraction only
T2		ACR-A7	2	Acropora	Adult	500 uL shield	T2 RNA	20250708	80	28.4	2272	rna extraction only
T3		ACR-A8	3	Acropora	Larvae	500 uL shield	T3 RNA	20250708	80	28.6	2288	rna extraction only
T4		ACR-A9	4	Acropora	Larvae	500 uL shield	T4 RNA	20250708	80	25.6	2048	rna extraction only
T5		ACR-A10	5	Acropora	Adult	800 ul shield	T5 RNA	20250708	80	34	2720	rna extraction only
T6		POC-A6	33	Pocillopora	Adult	500 uL shield	T5 RNA	20250708	80	67.6	5408	rna extraction only
T7		POC-A7	34	Pocillopora	Adult	500 uL shield	T6 RNA	20250708	80	39.4	3152	rna extraction only
T8		POC-R13	47	Pocillopora	Recruit	500 uL shield	T7 RNA	20250708	80	39.4	3152	rna extraction only
T9		POC-R15	49	Pocillopora	Recruit	500 uL shield	T8 RNA	20250708	80	59.4	4752	rna extraction only
T10		POC-R18	52	Pocillopora	Recruit	500 uL shield	T9 RNA	20250708	80	9.14	731.2	rna extraction only

Concentrations look good! In general RNA concentration is 20-50 ng/µL and I do not see any issues with a particular species or lifestage. I will move forward with processing the samples for sequencing.

Each day, I will thaw the samples on ice (n=10 per batch), bead beat, aliquot, extract RNA and DNA, and quantify RNA.

20250714

Today I extracted 10 samples for RNA and gDNA using the protocol linked above with no modifications.

I quantified using the Qubit after extractions.

RNA concentrations look good in general. The lowest concentration samples were the Pocillopora larvae, especially those from the 33C treatment.

seq_id	sample_id	tube_id	genus	lifestage	Treatment	rna.tube	dna.tube	date.extracted	extraction.batch	total.volume.ul	concentration.ng.ul	total.rna.ng
1	A7	A7	Acropora	Larvae	30	1 RNA	1 gDNA	20250714	1	80	22.8	1824
2	ACR-R22	24	Acropora	Recruit	30	2 RNA	2 gDNA	20250714	1	80	11.2	896
3	A10	A10	Acropora	Larvae	30	3 RNA	3 gDNA	20250714	1	80	24.8	1984
4	P15	P15	Pocillopora	Larvae	33	4 RNA	4 gDNA	20250714	1	80	4.6	368
5	P16	P16	Pocillopora	Larvae	33	5 RNA	5 gDNA	20250714	1	80	6.84	547.2
6	ACR-R24	26	Acropora	Recruit	27	6 RNA	6 gDNA	20250714	1	80	19.1	1528
7	P5	P5	Pocillopora	Larvae	27	7 RNA	7 gDNA	20250714	1	80	11.2	896
8	A6	A6	Acropora	Larvae	27	8 RNA	8 gDNA	20250714	1	80	20	1600
9	A2	A2	Acropora	Larvae	27	9 RNA	9 gDNA	20250714	1	80	24.4	1952
10	P6	P6	Pocillopora	Larvae	27	10 RNA	10 gDNA	20250714	1	80	10.6	848

I will proceed with another batch on Wednesday. Today, I bead beat and aliquoted samples 11-30 in preparation for future extractions.