S. pistillata RNA extraction with Zymo kit

Using the Zymo Quick-RNA Miniprep Kit on Stylophora pistillata samples across depths.

For this extraction I used the protocol of Federica Scucchia (fscucchia) with some adjustments.

This protocol was also checked with Oculina patagonica and with S. pistillata planulae (5-10 in one tube) - it works well with these samples.

Reagents preparation

  1. Add 96 mL 100% ethanol (104 mL 95% ethanol) to the 24 mL DNA/RNA Wash Buffer concentrate before use. DNA/RNA Wash Buffer included with D7003T (Mini Prep Plus Kit) is supplied ready-to-use and does not require the addition of ethanol prior to use. Check kit contents and instructions to confirm prep steps.
  2. Reconstitute the lyophilized (freeze-dried) DNase I as indicated on the vial prior to use. Mix by inversion. Store frozen aliquots.

Adult fragment sample preparation

  1. Sterilize the surface with 70% ethanol. Wear gloves.
  2. Check the concentration of the shield. If it’s Zymo shield, it comes in 1x and 2x concentrations. Final concentration of the shield with your sample should be 1x. If the sample is liquid, put 2x shield to the sample in 1:1 ratio. If the sample is solid, dilute 2x shield before putting the sample into it by adding DNAse/RNAse free molecular water in 1:1 ratio.
  3. Add 700 µl of DNA/RNA shield to a new 2 ml tube with a screw cap.
  4. Using sterilized clippers, clip off 2-3 small pieces of coral and put them in the 2 mL tube from step 3. The pieces shoud be small enough to fit into the tube. The DNA/RNA shield needs to cover the fragments. If you still have a lot of free space in the tube it’s better to add more shield so the samples are covered in the shield even when they are shaking during the transportation. If there is enough coral you can prepare spare tubes with the same sample.
  5. Sterilize clippers in between every sample.
  6. Leave samples in RNA shield on the bench (room temperature, <30°C) for 1-2 hours so the shield can penetrate into the tissue. Then put the samples in -20°C/-80°C for a prolonged storage.

Disruption of the coral skeleton

  1. Thaw samples on ice.
  2. Put 0.4-0.5 ml of 2 mm glass beads to the sample tube. Put samples into a Disruptor Genie for 1 minute at 2000 rpm. The amount of time for vortexing will depend on the coral skeletal structure and how easily the tissue separates from the skeleton. Too much vortexing can cause DNA/RNA degradation, but too little vortexing can result in minimal DNA/RNA yield.
  3. Transfer 345 µl of liquid into a new 1.5 ml tube. Save the tube with the skeleton fragment in -80°C freezer as a potential back-up in case the extraction doesn’t work.
  4. Proceed to RNA Extraction steps.

RNA Extraction

  1. Set up yellow DNA spin columns and collection tubes, label appropriately
  2. Warm elution liquid (RNase free water) to 65°C, get DNase I from freezer and put it on ice
  3. Add equal volume (345 µl) DNA/RNA lysis buffer to each sample tube
  4. Finger flick to mix tubes
  5. Add 700 µl (total volume) of sample gently to the yellow DNA spin column
  6. Centrifuge at 16,000 rcf (g) for 30 seconds
  7. Save the flow through from this step: transfer to a new 1.5mL tube
  8. Add equal volume (700 µl) 100% EtOH to the 1.5mL tubes containing the original yellow column flow through
  9. Vortex and spin down to mix
  10. Add 700 µl of that liquid to the green RNA spin columns
  11. Centrifuge at 16,000 rcf (g) for 30 seconds
  12. Discard flow through (Zymo kit waste)
  13. Add 700 µl to the green RNA spin columns (the rest from the 1.5mL RNA tubes)
  14. Centrifuge at 16,000 rcf (g) for 30 seconds
  15. Discard flow through (Zymo kit waste)
  16. Add 400 µl DNA/RNA Wash Buffer gently to each green RNA column
  17. Centrifuge at 16,000 rcf (g) for 30 seconds
  18. Discard flow through (Zymo kit waste)
  19. Make DNase I treatment master mix:
    • 75µl DNA Digestion buffer x # of samples
    • 5µl DNase I x # of samples
  20. Add 80 µl DNase I treatment master mix directly to the filter of the green RNA columns
  21. Incubate at room temp for 15 minutes
  22. Add 400 µl DNA/RNA Prep Buffer gently to each column
  23. Centrifuge at 16,000 rcf (g) for 30 seconds
  24. Discard flow through (Zymo kit waste)
  25. Add 700 µl DNA/RNA Wash Buffer gently to the green RNA spin columns
  26. Centrifuge at 16,000 rcf (g) for 30 seconds
  27. Discard flow through (Zymo kit waste)
  28. Add 400 µl DNA/RNA Wash Buffer gently to the green RNA spin columns
  29. Centrifuge at 16,000 rcf (g) for 2 minutes
  30. Discard flow through (Zymo kit waste)
  31. Transfer green columns to new 1.5mL microcentrifuge tubes labelled appropriately
  32. Add 37µl warmed DNase/RNase free water to each green RNA column by dripping slowly directly on the filer
  33. Incubate at room temp for 5 minutes
  34. Centrifuge at 16,000 rcf (g) for 30 seconds
  35. Add 30µl warmed DNase/RNase free water to each green RNA column by dripping slowly directly on the file for a final elution volume of 67µl
  36. Incubate at room temp for 5 minutes
  37. Centrifuge at 16,000 rcf (g) for 30 seconds
  38. Put 1.5mL tubes on ice afterwards, put 7µl aliquot into 0.2 ml PCR tubes to save QC for Nanodrop and Tape Station to avoid freeze-thaw of your stock sample, put 20µl aliquot into a new 1.5ml tube for metagenomics.
  39. Store all tubes in the -80°C

RNA Quality

If RNA quantity is sufficient (for sequencing you need to have a total of 500 ng per sample), determine RNA quality using the Tape Station. “Good” RNA should have a RIN above 8.0 and form two distinct peaks at the 18S and 28S locations.

This protocol was created on 06.11.24.

Written on November 6, 2024