RNA Re-extractions for Time Series Project, August 29, 2025

Continuation of Natalie’s amazing work extracting these samples, latest post here

Protocol Link

Re-extraction of 5 Montipora capitata samples from the Time Series experiment done in June and July of 2025. These samples did not yield enough RNA to be measured by HS RNA Qubit so re-extraction was needed.

I made several adjustments to the protocol to try to maximize yield and minimize mucus carryover.

Samples

The sample IDs are MON 1 H1, MON 3 H1, MON 24 H1, MON 24 C2, and MON 120 C2.

Notes

• For sample prep bead beat was used in the original tube and vortexed for an additional 1 minute.
• Took 2X volume out of the DNA/RNA shield tube to double biomass input into extraction (especially because the shield in these tubes has been refilled, so much of the tissue was already lysed into DNA/RNA shield that had been removed in previous extractions)
• 600 uL of new DNA/RNA shield was added to sample tubes after the 600 uL was taken out for extraction for storage.
• Adjusted protocol for 2X volume accordingly: added 60 uL ProK digestion buffer and 30 uL ProK
• Performed a heated proteinase K digestion after adding the above and inverting 3X.
  • Digested at 55 ºC on the thermomixer, at 300 rpm, for 10 minutes
• Then spun down to pellet debris for 10 minutes at 9,000 rcf, at 4 ºC
• Then moved supernatant to new tube and added equal volume DNA/RNA lysis buffer. This had to be run through the DNA column in two separate additions of 700 uL
• Flow through from each spin was kept and moved into a new 1.5 mL tube
• To each of those flow-through tubes, 700 uL ethanol was added (worked one tube at a time) and mixed thoroughly (pipette up and down 10 times)
• Then 700 uL of this mixture was added to the RNA column and spun
• Then repeated with the remaining 700 uL in that tube
• Then did the process again for the second 1.5 mL tube of flow through
• Rest of extraction performed as written (except changes below)
• Eluted RNA to 80 uL of RNAse/DNAse free water warmed to 37 ºC (2 additions of 40 uL)
  • After the second elution, the entire eluate was reapplied to the column to get out any last RNA
  • DNA was not extracted
• 2 uL were used for Qubit and 8 uL were used for gel

Qubit Results

• Used Broad range dsDNA and RNA Qubit Protocol HERE
• All samples read twice, standard only read once

HS RNA Standards: 49.50 (S1) and 1038.22 (S2)

colony_id	Species	RNA_QBIT_1	RNA_QBIT_2	RNA_QBIT_AVG
1 H1	Montipora capitata	3.76	3.83	3.795
3 H1	Montipora capitata	3. 38	3.4	3.39
24 H1	Montipora capitata	14.8	14.8	14.8
24 C2	Montipora capitata	10.1	10.7	10.4
120 C2	Montipora capitata	12.9	12.9	12.9

• RNA samples in -80 freezer

DNA and RNA Quality Check gel

Ran at 60 volts for 60 minutes