RNA Extraction Log for 2023 Moorea Project

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.

Batch 1:

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.

20250716

Today I extracted 20 samples (2 batches of 10) for RNA and gDNA using the protocol linked above with no modifications. Aakriti completed gDNA extraction steps following lysis. All samples stored in -80°C.

I quantified using the Qubit after extractions.

RNA concentrations look good in general. The lowest concentration samples were the Pocillopora larvae, consistent with previous extractions. The facility requires >200 ng total RNA, so we are above this limit so far.

Batch 2:

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
11 POC-R19 53 Pocillopora Recruit 33 11 RNA 11 gDNA 20250716 2 80 57 4560
12 A4 A4 Acropora Larvae 27 12 RNA 12 gDNA 20250716 2 80 27.8 2224
13 P12 P12 Pocillopora Larvae 30 13 RNA 13 gDNA 20250716 2 80 6.66 532.8
14 POC-R11 45 Pocillopora Recruit 27 14 RNA 14 gDNA 20250716 2 80 32.2 2576
15 P9 P9 Pocillopora Larvae 30 15 RNA 15 gDNA 20250716 2 80 7.48 598.4
16 P18 P18 Pocillopora Larvae 33 16 RNA 16 gDNA 20250716 2 80 8.68 694.4
17 POC-R28 62 Pocillopora Recruit 27 17 RNA 17 gDNA 20250716 2 80 22.2 1776
18 A17 A17 Acropora Larvae 33 18 RNA 18 gDNA 20250716 2 80 32.2 2576
19 P3 P3 Pocillopora Larvae 27 19 RNA 19 gDNA 20250716 2 80 6.04 483.2
20 A9 A9 Acropora Larvae 30 20 RNA 20 gDNA 20250716 2 80 32.2 2576

Batch 3:

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
21 A16 A16 Acropora Larvae 33 21 RNA 21 gDNA 20250716 3 80 48.2 3856
22 P4 P4 Pocillopora Larvae 27 22 RNA 22 gDNA 20250716 3 80 8.08 646.4
23 ACR-R28 30 Acropora Recruit 30 23 RNA 23 gDNA 20250716 3 80 8.88 710.4
24 A3 A3 Acropora Larvae 27 24 RNA 24 gDNA 20250716 3 80 42.6 3408
25 POC-R26 60 Pocillopora Recruit 30 25 RNA 25 gDNA 20250716 3 80 7.22 577.6
26 P14 P14 Pocillopora Larvae 33 26 RNA 26 gDNA 20250716 3 80 7.92 633.6
27 ACR-R29 31 Acropora Recruit 27 27 RNA 27 gDNA 20250716 3 80 27 2160
28 ACR-R18 20 Acropora Recruit 30 28 RNA 28 gDNA 20250716 3 80 17.5 1400
29 ACR-R25 27 Acropora Recruit 30 29 RNA 29 gDNA 20250716 3 80 29.2 2336
30 P10 P10 Pocillopora Larvae 30 30 RNA 30 gDNA 20250716 3 80 7.5 600

We are using the following equipment:

  • Eppendorf Centrifuge 5424
  • Qubit High Sensitivity RNA 3.0 Fluoreometer and reagents
  • Bullet Blender bead beater Gold +

Batches 1-3 used the following kit: Zymo Quick RNA DNA MiniPrep Plus Kit (cat no. D7003, Lot no. 225465).

I will proceed with another batch on Friday using a new kit (Lot no. 225465). I prepped the reagents today.

20250718

Today I extracted 14 samples (1 batch of 14) for RNA and gDNA using the protocol linked above with no modifications. Aakriti completed gDNA extraction steps following lysis. All samples stored in -80°C.

I quantified using the Qubit after extractions.

RNA concentrations look good in general. The lowest concentration samples were the Pocillopora larvae, consistent with previous extractions.

Batch 4:

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
31 POC-R21 55 Pocillopora Recruit 27 31 RNA 31 gDNA 20250718 4 80 22.4 1792
32 ACR-R15 17 Acropora Recruit 33 32 RNA 32 gDNA 20250718 4 80 19.4 1552
33 POC-R14 48 Pocillopora Recruit 33 33 RNA 33 gDNA 20250718 4 80 46.6 3728
34 P7 P7 Pocillopora Larvae 30 34 RNA 34 gDNA 20250718 4 80 6.98 558.4
35 ACR-R27 29 Acropora Recruit 27 35 RNA 35 gDNA 20250718 4 80 24.6 1968
36 ACR-R12 14 Acropora Recruit 33 36 RNA 36 gDNA 20250718 4 80 12.7 1016
37 A8 A8 Acropora Larvae 30 37 RNA 37 gDNA 20250718 4 80 38.2 3056
38 P8 P8 Pocillopora Larvae 30 38 RNA 38 gDNA 20250718 4 80 7.42 593.6
39 A15 A15 Acropora Larvae 33 39 RNA 39 gDNA 20250718 4 80 28.4 2272
40 A13 A13 Acropora Larvae 33 40 RNA 40 gDNA 20250718 4 80 28.6 2288
41 A12 A12 Acropora Larvae 30 41 RNA 41 gDNA 20250718 4 80 35.8 2864
42 ACR-R19 21 Acropora Recruit 33 42 RNA 42 gDNA 20250718 4 80 15.7 1256
43 ACR-R17 19 Acropora Recruit 27 43 RNA 43 gDNA 20250718 4 80 16.7 1336
44 ACR-R14 16 Acropora Recruit 30 44 RNA 44 gDNA 20250718 4 80 17.9 1432

Batch 4 used a new kit with the following information: Zymo Quick RNA DNA MiniPrep Plus Kit (cat no. D7003, Lot no. 225465).

20250721

Today I extracted 10 samples (1 batch of 10) for RNA and gDNA using the protocol linked above with no modifications. Aakriti completed gDNA extraction steps following lysis. All samples stored in -80°C.

I quantified using the Qubit after extractions.

RNA concentrations look good in general. The lowest concentration samples were the Pocillopora larvae, consistent with previous extractions.

Batch 5:

seq_id box sample_id tube_id genus lifestage preservation Treatment rna.tube dna.tube date.extracted extraction.batch total.volume.ul concentration.ng.ul total.rna.ng notes
45 Box 4 P13 P13 Pocillopora Larvae 250 uL shield 33 45 RNA 45 gDNA 20250721 5 80 8.34 667.2  
46 RNA Box 1 ACR-R30 32 Acropora Recruit 500 uL shield 33 46 RNA 46 gDNA 20250721 5 80 32.4 2592  
47 RNA Box 2 POC-R12 46 Pocillopora Recruit 500 uL shield 30 47 RNA 47 gDNA 20250721 5 80 26.8 2144  
48 Box 4 A14 A14 Acropora Larvae 500 uL shield 33 48 RNA 48 gDNA 20250721 5 80 51.4 4112  
49 Box 4 P2 P2 Pocillopora Larvae 250 uL shield 27 49 RNA 49 gDNA 20250721 5 80 10 800  
50 RNA Box 1 ACR-R13 15 Acropora Recruit 500 uL shield 33 50 RNA 50 gDNA 20250721 5 80 22.6 1808  
51 Box 4 P1 P1 Pocillopora Larvae 250 uL shield 27 51 RNA 51 gDNA 20250721 5 80 6.06 484.8  
52 Box 4 P17 P17 Pocillopora Larvae 250 uL shield 33 52 RNA 52 gDNA 20250721 5 80 5.34 427.2  
53 RNA Box 1 ACR-R23 25 Acropora Recruit 500 uL shield 27 53 RNA 53 gDNA 20250721 5 80 26.4 2112  
54 RNA Box 1 ACR-R11 13 Acropora Recruit 500 uL shield 27 54 RNA 54 gDNA 20250721 5 80 13.8 1104  

20250722

Today I extracted 10 samples (1 batch of 10) for RNA and gDNA using the protocol linked above with no modifications. Aakriti completed gDNA extraction steps following lysis. All samples stored in -80°C.

I quantified using the Qubit after extractions.

RNA concentrations look good in general. The lowest concentration samples were the Pocillopora larvae, consistent with previous extractions.

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
55 POC-R24 58 Pocillopora Recruit 30 55 RNA 55 gDNA 20250722 6 80 44.8 3584
56 A18 A18 Acropora Larvae 33 56 RNA 56 gDNA 20250722 6 80 21 1680
57 A5 A5 Acropora Larvae 27 57 RNA 57 gDNA 20250722 6 80 28 2240
58 P11 P11 Pocillopora Larvae 30 58 RNA 58 gDNA 20250722 6 80 10.5 840
59 ACR-R26 28 Acropora Recruit 30 59 RNA 59 gDNA 20250722 6 80 23.2 1856
60 A1 A1 Acropora Larvae 27 60 RNA 60 gDNA 20250722 6 80 31.2 2496
61 POC-R30 64 Pocillopora Recruit 33 61 RNA 61 gDNA 20250722 6 80 33.2 2656
62 POC-R16 50 Pocillopora Recruit 33 62 RNA 62 gDNA 20250722 6 80 5.18 414.4
63 A11 A11 Acropora Larvae 30 63 RNA 63 gDNA 20250722 6 80 34.2 2736
64 ACR-R16 18 Acropora Recruit 33 64 RNA 64 gDNA 20250722 6 80 14 1120

I prepared a box and will ship the samples out on Monday with overnight delivery.

Written on July 21, 2025