Coral RNA Extraction Protocol for 2023 Moorea Project

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

RNA Extraction Protocol

This protocol is based on Jill’s extraction protocol. This post details RNA extraction protocols for samples taken during the 2023 Symbiotic Exchange project.

Samples

Samples include larvae preserved in RNA/DNA shield as well as clippings from recruit corals preserved in RNA/DNA shield.

Materials

  • Zymo Quick-DNA/RNA Miniprep Plus Kit and Protocol
  • Tris-Ethylenediaminetetraacetic acid (EDTA) 1X buffer for DNA elution
  • Heating block capable of heating to 70°C
  • Centrifuge and rotor capable of spinning at 15,000 rcf
  • Sample tube freezer boxes (1 for RNA and 1 for DNA)
  • Plastics
    • 3 1.5 mL microcentrifuge tubes per sample
    • 1 PCR tube per sample
    • 1 Qubit tube per sample
    • 1 5 mL tubes per sample
  • Beads for bead beating RPI Glass disruption beads

Protocol

Sample prep and homogenization: Larvae

  • Thaw samples in an ice bucket.

  • Spin down tubes so there are no bubbles in lid

  • Add ~50 uL of glass beads (0.5mm) into sample tubes (enough to cover the bottom of the tube)

  • Bead beat tubes at high speed for 2 minutes.

  • Spin down in mini centrifuge for 1 minute

  • Remove 500 uL of liquid sample from the original sample tube into a new tube to proceed with extraction. Save the leftover sample + beads at -80°C.

Sample prep: Recruit fragments

  • Thaw samples in an ice bucket.

  • Depending on the color of the shield pigementation, bead beating may be needed. If the shield is pale, bead beat the sample.

  • Beat beat tubes at high speed for 2 minutes.

  • Spin down in mini centrifuge for 1 minute

  • Remove 500 uL of liquid sample from the original sample tube into a new tube to proceed with extraction. Save the leftover sample + beads at -80°C.

Lysis

  • Add 50 uL of Proteinase K digestion buffer (10:1 ratio of sample:digestion buffer) and 25 uL of Proteinase K (2:1 ratio of digestion buffer:Proteinase K) to each sample.

  • Vortex. Incubate on counter for 30 min. Spin down.

  • Add 575 uL of lysis buffer (1:1 ratio of sample:lysis buffer) to each sample. The volume in each tube should be 1150 uL.

  • Vortex and spin down.

  • Transfer 700 uL of the sample into the corresponding yellow DNA spin column. Spin for 30 seconds at 16000 rpm.

  • Move the liquid collected in the collection tube into a labeled 5 mL tube. This is your RNA!

  • Transfer the remainder of the sample (~450 uL) into the yellow DNA spin column. Spin for 30 seconds at 16000 rpm.

  • Move the liquid collected in the collection tube into the labeled 5 mL tube to combine with the liquid from the last spin. This is your RNA! Set this aside until RNA extraction steps.

  • The DNA remains on the filter of the yellow DNA spin column.

DNA extraction

  • Move yellow DNA column to new collection tube and discard the old one.

  • Add 400 uL of prep buffer. Cenrifuge for 30 seconds at 16000 rpm. Discard liquid.

  • Add 700 uL of wash buffer. Cenrifuge for 30 seconds at 16000 rpm. Discard liquid.

  • Add 400 uL of wash buffer. Cenrifuge for 2 minutes at 16000 rpm. Discard liquid.

  • Centrifuge columns dry for 2 minutes at 16000 rpm.

  • Move yellow DNA spin column into final labeled gDNA 1.5 mL tube (“ID gDNA Date”).

  • Add 40 µL of warm Tris to spin column. Let columns sit for 5 minutes at room temperature. Spin for 30 seconds at 16000 rpm. DO THIS TWICE! Total elution volume = 80 µL

  • Store the gDNA at -20°C.

RNA extraction

  • Add 1150 uL of 100% EtOH (1:1 ratio of sample:ethanol) to the 5 mL tube with the RNA. Pipette up and down to mix.

  • Transfer 700 uL of sample into the green RNA spin column. Spin for 30 seconds at 16000 rpm. Discard liquid.

  • Repeat the above step until all liquid has been moved from the 5 mL tube.

  • Add 400 uL of wash buffer. Centrifuge for 30 seconds at 16000 rpm. Discard liquid.

  • Make DNase rxn mix
    • To make DNase rxn mix:
      • DNA digestion buffer: 75 µL x ___ samples = ___ buffer needed
      • DNase I: 5 µL x ___ samples = ___ DNase I needed
    • DNase I is stored at -20°C
    • Combine in 1.5 mL tube and invert gently to mix

  • Add 80 uL of DNase mix to each spin column. Incubate at room temperature for 15 minutes.

  • Add 400 uL of prep buffer. Centrifuge for 30 seconds at 16000 rpm. Discard liquid.

  • Add 700 uL of wash buffer. Centrifuge for 30 seconds at 16000 rpm. Discard liquid.

  • Add 400 uL of wash buffer. Centrifuge for 2 minutes at 16000 rpm. Discard liquid.

  • Repeat the wash. Add 400 uL of wash buffer. Centrifuge for 2 minutes at 16000 rpm. Discard liquid.

  • Centrifuge columns dry for 2 minutes at 16000 rpm.

  • Move green RNA spin column into final RNA 1.5 mL tube (“ID RNA”).

  • Add 40 µL of warmed DNA/RNA free water to spin column. Let columns sit for 5 minutes. Spin for 30 seconds at 16000 rpm. DO THIS TWICE! Total elution volume = 80 µL

  • Aliquot 12 µL into one PCR tubes for QC. Store the rest of the RNA at -80°C.

QC

QC using Qubit.

Qubit

Prep for sending for sequencing

  • Metadata sheet and labeled tubes

  • Send around 70 µL (send the whole tube)

Tips and tricks

  • Start with a couple to troubleshoot

  • Can work our way up to doing 10-12 at a time

  • Prep tubes and boxes before starting

  • Print out protocol

  • Clean everything before starting with ethanol and RNAase spray

  • Avoid touching anything and be super sterile

  • Every 5 min do RNAase and ethanol washes on gloves and surfaces

Written on May 21, 2025