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 safe lock tube, 2 regular tubes)
    • 1 5 mL tubes per sample
  • Beads for bead beating RPI Glass disruption beads
  • 70% ethanol
  • 10% bleach
  • Elmininase
  • Tweezers (for transfering samples if needed in first section)

Protocol

Sample prep and homogenization: Larvae

  • Obtain dry ice for the Bullet Blender bead beater.

  • Thaw samples in an ice bucket.

  • Transfer sample to a labeled 1.5 mL Safe-Lock tube.

  • Add more RNA/DNA shield to the sample to bring total volume to 1000 µL.

  • 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)

  • Place Safe-Lock tubes in specified Bullet Blender adapters. Fill Bullet Blender with dry ice.

  • Bead beat tubes at high speed for 2 minutes.

  • Spin down in mini centrifuge for 1 minute.

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

Sample prep: Recruit fragments

  • Obtain dry ice for the Bullet Blender bead beater.

  • Thaw samples in an ice bucket.

  • Transfer sample to a labeled 1.5 mL Safe-Lock tube.

  • Add more RNA/DNA shield to the sample to bring total volume to 1000 µL.

  • Place Safe-Lock tubes in specified Bullet Blender adapters. Fill Bullet Blender with dry ice.

  • Bead beat tubes at high speed for 2 minutes.

  • Beat beat tubes at high speed for 2 minutes.

  • Spin down in mini centrifuge for 1 minute.

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

Lysis

  • 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 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

  • Warm tris to 70°C in a heating block.

  • 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 (70°C) 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

  • Warm RNA/DNA free water to 56°C in a warming block.

  • 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.

  • 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 (56°C) to spin column. Let columns sit for 5 minutes. Spin for 30 seconds at 16000 rpm. DO THIS TWICE! Total elution volume = 80 µL

  • Proceed directly to QC. Store the RNA at -80°C if not proceeding to QC.

QC

QC using the Qubit High Sensitivity RNA assay.

  • Remove kit standards from the fridge.

  • Set up the required number of Qubit 0.5 mL tubes (n=1 per sample with 2 additional for the 2 standards). Label teh lids of the Qubit tubes with the sample numbers and “S1”/”S2” for the standards.

  • Prepare Qubit working solution. The final volume is 200 µL. Samples will have 1 µL of sample and standards will have 10µL of standard.

    • Samples: 199 µL x ___ samples = ___ solution needed
    • Standards: 190 µL x ___ samples = ___ solution needed
    • Total = ___ solution needed + 10% = __ total solution
    • HS Qubit Reagent total volume = ___ total solution * (1/200) = ____ HS Reagent Volume

    • HS Qubit buffer total volume = total __ solution needed - ___ HS Reagent volume

    • For example:

    • Samples: 199 µL x 10 samples = 1990 µL solution needed
    • Standards: 190 µL x 2 samples = 380 µL solution needed
    • Total = 2370 µL solution needed + 10% = 2607 µL total solution
    • HS Qubit Reagent total volume = 2607 µL total solution * (1/200) = 13.04 µL HS Reagent Volume
    • HS Qubit buffer total volume = total 2607 µL solution needed - 13.04 HS Reagent volume = 2594 µL buffer

  • Add 190 µL of working solution to the S1 and S2 standard tubes.

  • Add 10 µL (exactly!) of Standard 1 to the S1 tube and 10 µL of Standard 2 to the S2 tube.

  • Vortex.

  • Add 199 µL to all sample Qubit tubes.

  • Add 1 µL of sample to respective Qubit sample tubes.

  • Vortex.

  • Incubate tubes at room temperature for 2 min.

  • Read standards

    • On the Qubit (3.0), press “RNA”, then “RNA: High Sensitivity”. Press “Read standards”.
    • Insert the S1 tube. Press “Read standard”. Remove.
    • Insert the S2 tube. Press “read standard”. Remove.
    • Record calibration information.

  • Run samples

    • Select the sample volume and units (200 µL total, 199 µL working solution, 1 µL sample)
    • Select ng/µL units.
    • Insert a sample tube and press “Read tube”. Remove the tube when done.
    • Record the data - include the top value (concentration) and the bottom value (dilution concentration).
    • Repeat to read all samples. Record the order in which you read all samples.

Prep for sending for sequencing

  • Metadata sheet and labeled tubes

  • Send around 70 µL (send the whole tube)

  • Package with dry ice

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 June 5, 2025