RNA and DNA Extractions for ENCORE Project, October 19, 2022

Protocol Link

Extraction of 2 samples from ENCORE Project. These samples were collected in Bermuda in August 2022 for the ENCORE TPC project. There are 160 samples total from the following 4 species: Diploria labyrinthiformis (DLAB), Montastraea cavernosa (MCAV), Madracis decactis (MDEC), and Porites astreoides (PAST)

Samples

Testing different extraction methods for PAST samples.

• Test 1: Using less of the sample DNA/RNA shield (100 uL) in the initial ProK digestion
• Test 2: Bead-beating using Kevin Wong’s protocol here:

Sample Preparation and Digestion

1. Add 0.25 mL of glass beads (0.5mm) and add 500 μl of RNA/DNA shield into each empty centrifuge tube.
2. Take samples out from -80 °C on dry ice.
3. Using sterile clippers, add 0.25mm of tissue into the centrifuge tube with beads and RNA/DNA shield.
4. Vortex at max speed for 2 minutes.
5. Remove 400 μl of the supernatant and transfer to a new centrifuge tube.
6. Centrifuge at 9000 rcf for 3 minutes.
7. Transfer 300 μl supernatant to a new centrifuge tube and discard the pellet.
8. Add 30 μl of Proteinase K digestion buffer (10:1 ratio of sample:digestion buffer), and 15 μl of Proteinase K (2:1 ratio of digestion buffer:Proteinase K) to each sample.
9. Vortex and spin down.
10. Add 345 μl of lysis buffer.
11. Continue with DNA and RNA extraction protocol

Notes

• Followed protocol exactly.

• I did two extractions per tube. For one, of those extractions, I took 100 uL of the DNA/RNA shield from the sample tube and added 200 uL of clean DNA/RNA shield before proceeding with the extraction as written.

• For the second extraction, I followed Kevin’s bead-beating protocol as written at the end of my last notebook entry. I added 0.25 mL of glass 0.5mm beads to a clean screw-top 1.5 mL tube and added 200 of the sample DNA/RNA shield with 200 of clean DNA/RNA shield. Then, I bead-beat using a vortexer on high speed for 2 minutes. Then, I removed 400 uL of the supernatant (to achieve this, I needed to add another 100 uL of clean DNA/RNA shield, so in the future I will add 300 uL of clean DNA/RNA shield at the beginning instead of 200 uL) into a clean 1.5 mL tube and spun down at 9,000 rcf for 3 minutes. Then, I removed 300 uL of the supernatant into a clean tube and proceeded with the ProK digestion as written in the protocol.

• Throughout the RNA extraction, the filters of two bead-beat samples were pigmented. So were the final RNA for those tubes.

Qubit Results

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

DNA Standards: 190.31 (S1) & 21981.24 (S2)

colony_id	Species	Temp.Cat	DNA_QBIT_1	DNA_QBIT_2	DNA_QBIT_AVG
Past-A6	Porites astreoides	26	2.34	2.26	2.3
Past-A6, bead-beat	Porites astreoides	26	5.38	5.26	5.32
Past-C1	Porites astreoides	29	2.48	2.38	2.43
Past-C1, bead-beat	Porites astreoides	29	11.1	10.9	11

RNA Standards: 417.20 (S1) & 9353.57 (S2)

colony_id	Species	Temp.Cat	RNA_QBIT_1	RNA_QBIT_2	RNA_QBIT_AVG
Past-A6	Porites astreoides	26	nd	nd	0
Past-A6, bead-beat	Porites astreoides	26	nd	nd	0
Past-C1	Porites astreoides	29	nd	nd	0
Past-C1, bead-beat	Porites astreoides	29	11.4	11.2	11.3

DNA and RNA Quality Check: Gel, using Kristina’s gel protocol at the bottom of this page.

Next steps

• As mentioned in the “Notes” section above, if I bead-beat again I will use 300 uL of clean DNA/RNA shield (or a different combination of clean and sample DNA/RNA shield) to achieve a final volume of 500 uL in the bead-beating tube.

• I am still disappointed by the quantity and quality of RNA… Next will try to heat the sample during ProK digestion to 55 ºC for 2-3 hours, as used in Erin E. Chille’s protocol.

• RNA is still pigmented, especially from the bead-beat samples which also had colored RNA filters. Maybe bead-beating is still an option with less of the sample DNA/RNA shield.

Here is an image of the two PAST-A6 final RNA tubes, with the bead-beat sample on the right.

Here is an image of the two PAST-C1 final RNA tubes, with the bead-beat sample on the right.