RNA and DNA Extractions for ENCORE Project, October 21, 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 and heating to 55ºC for 60 minutes during the ProK digestion.
• Test 2: Bead-beating using a modified version of 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. Add 200 uL of the sample DNA/RNA shield to the tube
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. Then I heated these at 55ºC for 60 minutes during the ProK digestion step.

• 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 the coral fragment with 200 of the sample DNA/RNA shield with 500 (on 10/19/22 I only added 200 uL) 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 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.

• The heated to 55 ºC extraction for PAST-D1 had issues:

  • The pellet was very loose after spinning down after the ProK/heating step.
  • The supernatant was still very viscous here, and was not mixing well with the lysis buffer.
  • The RNA filter was pigmented throughout the extraction.
  • The RNA was pigmented at the end of the extraction.

Qubit Results

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

DNA Standards: 189.15 (S1) & 23503.13 (S2)

colony_id	Species	Temp.Cat	DNA_QBIT_1	DNA_QBIT_2	DNA_QBIT_AVG
Past-C6, heated	Porites astreoides	26	nd	nd	0
Past-C6, bead-beat	Porites astreoides	26	3.00	2.92	2.96
Past-D1, heated	Porites astreoides	29	2.18	2.08	2.13
Past-D1, bead-beat	Porites astreoides	29	7.98	7.80	7.89

RNA Standards: 417.59 (S1) & 9757.72 (S2)

colony_id	Species	Temp.Cat	RNA_QBIT_1	RNA_QBIT_2	RNA_QBIT_AVG
Past-C6, heated	Porites astreoides	26	nd	nd	0
Past-C6, bead-beat	Porites astreoides	26	nd	nd	0
Past-D1, heated	Porites astreoides	29	nd	nd	0
Past-D1, bead-beat	Porites astreoides	29	nd	nd	0

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

Next steps

OKay. Qubit RNA looks bad, but there are bands for C6, bead-beat!!! So, maybe next I will try to stick with bead-beating but with even less DNA/RNA shield from the sample tube added. Maybe by just beating the fragment with clean DNA/RNA shield this will further reduce the mucus that enters the extraction.