Adult Coral RNA and DNA Extraction using the Zymo Quick-DNA/RNA™ Miniprep Plus Kit

This protocol is based off of the base Zymo protocol, which was adjusted for Putnam Lab needs by Emma L. Strand, Dr. Kevin H. Wong (and here), and Erin E. Chille. Kristina Terpis (Porites sp. example) modified this protocol for coral clippings stored in 1mL of Zymo DNA/RNA shield and kept at -80 ºC.

Goal: to extract high-quality gDNA and total RNA from adult tissue of diverse stony coral species.

Materials and Equipment

  • Zymo Quick-DNA/RNA Miniprep Plus Kit HERE Protocol Booklet
  • Tris-Ethylenediaminetetraacetic acid (EDTA) 1X buffer for DNA elution
  • Heating block capable of heating to 70ºC
  • Centrifuge and rotor capable of spinning at 15,000 rcf
  • Plastics: 5 1.5 mL microcentrifuge tubes per sample, 2 PCR tubes per sample, several 1000 uL, 200 uL, and 20 uL pipette tips per sample.

First time opening kit: Reagent Preparation

  1. Add 96 mL 100% ethanol to the 24 mL DNA/RNA Wash Buffer concentrate before use. Mark clearly on the bottle that Ethanol was added. 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. For 250U of DNAse I, add 275 uL of DNAse/RNAse free water (provided). For 50U of DNAse I, add 55 uL of DNAse/RNAse free water (provided). Mix by gentle inversion. Check kit contents and instructions to confirm prep steps. Separate into 3-4 aliquots to minimize freeze-thaw and store at -20 ºC.
  3. Reconstitute the lyophilized (freeze-dried) Proteinase K as indicated on the vial prior to use. For 20g Proteinase K, add 1040 uL Proteinase K Storage Buffer. For 5 mg Proteinase K, add 260 uL Proteinase K Storage Buffer. Mix by vortexing. Check kit contents and instructions to confirm prep steps. Store tube at -20 ºC, no need to aliquot.

Sterilizing working area to maintain an RNAse-free environment

Clean bench with clean paper towels (spray solution, wipe down) in the following order:

  1. 10% bleach solution
  2. DI water
  3. 70% ethanol
  4. RNAse cleaner (spray bottle)

Clean pipettes, tip boxes, and the controls on the heating block and centrifuge by squirting 70% ethanol on a paper towel and wiping them down. Repeat with RNAse cleaner. Wipe down gloves with 70% ethanol and RNAse cleaner. Do not spray solutions directly on the equipment.

Sample preparation: Adult Fragment Tissue Sample in Zymo DNA/RNA Shield

This protocol utilizes coral clippings stored in 1mL of Zymo DNA/RNA shield and kept at -80 ºC. Thaw samples to room temperature on benchtop. On a clean work surface (see above), stand up the tubes in numerical order and photograph to record qualitative qualities such as buffer color and amount of tissue in tube.

Notes on sample preparation

This protocol will start by taking 300 uL of the DNA/RNA shield from the sample tube into a clean 1.5 mL tube and performing a Proteinase K digestion in a new tube. However, there are several adjustments that can be made at this step.

  • If the DNA/RNA shield looks very dark (ex: this occurs a lot with highly pigmented species such as Porites spp.), only take 150 or 100 uL of the DNA/RNA shield from the sample tube into the new tube and dilute with 150 uL or 200 uL of clean DNA/RNA shield (provided in kit). Proceed as directed.

  • If the extraction is not yielding enough DNA or RNA and the DNA/RNA shield looks very light-colored, it could be that the lytic properties of the DNA/RNA shield did not break up the tissue enough to yield enough DNA and RNA from the buffer alone. In this case, you could bead beat this sample using the protocols described by other lab members Emma L. Strand, Dr. Kevin H. Wong, and Kristina Terpis. This bead beating step could be done by adding beads to the original sample tube, or by transferring an aliquot (300 uL) of the DNA/RNA shield from the sample tube along with a clipping of the tissue fragment into a new bead tube. Add 500-700 uL of clean DNA/RNA shield to make up 800 uL - 1 mL for bead-beating and bead beat for 1-2 minutes at maximum speed. Then, centrifuge briefly and take an aliquot of 300 uL from this homogenized mixture into a clean 1.5 mL tube. Proceed as directed.

Note: I have used this protocol for samples in RNAlater as follows:

  1. Thawed samples on ice.
  2. With sterilized forceps (10% bleach, DEPC water, 70% ethanol, air-dried), I removed one small (5mm x 5mm) fragment from the tube, in which most tubes had 5-6 small fragments of this size. I sterilized forceps between each sample.
  3. I briefly transferred this fragment to a clean 1.5 mL tube on ice or used a kimwipe to tap off any excess RNAlater and then immediately transferred the fragment to a 1.5 mL screw-cap tube with 1000 uL DNA/RNA shield and 0.25 mL of 0.5mm glass beads.
  4. Bead beat for 1-2 minutes on the vortex at max speed. I started off with one minute for all samples but added a minute of bead-beating for samples if the liquid still looked very light-colored after one minute of homogenization.
  5. Briefly spin down and remove 400 uL of supernatant into a clean tube. Spin for 3 mins at 9,000 rcf.
  6. Put original samples and bead tubes back in -80 ºC freezer.
  7. Remove 300 uL of the supernatant into a new tube and continue on with the protocol below (from the Pro K step) as written.

Protocol Steps

Proteinase K Digestion

  • On a clean work surface (see above), stand up the tubes in numerical order and photograph to record qualitative qualities such as buffer color and amount of tissue in tube.
  • Label a clean 1.5 mL microcentrifuge tube for each sample with sample number. This is a temporary tube, so you can use a shortened version of the sample number if desired. Make sure your labeling is clear. Ethanol is used in this protocol and can very easily rub off permanent marker writing on tubes.
  1. Invert all sample tubes 3 times to mix buffer well before pipetting out supernatant.
  2. Into a clean 1.5 mL microcentrifuge tube, transfer 300 uL of the supernatant from the sample tube (see notes above).
    • Add: 30 µl of PK digestion buffer to each sample tube (1:10 ratio of PK Digestion Buffer:Sample)
    • Add: 15 µl Proteinase K to each sample tube (1:2 ratio of Proteinase K:PK Digestion Buffer).
    • Invert 3x to mix.
  3. Incubate for 15 minutes at room temperature.
  4. Place original sample tubes back in the freezer box (in -80 ºC) that they came from. Always keep these as backup.

Lysis and Column Transfer

  • Turn on heating block to 70 ºC and fill a 1.5 mL microcentrifuge tube with Tris-EDTA (see materials, we use this solution as is) for DNA elution. You will need 100 uL for each sample, so aliquot 1 mL per 10 samples. Place this tube in the heating block so it will be at 70 ºC when you are ready to elute.
  • Label two clean 1.5 mL microcentrifuge tubes for each sample. For one tube per sample, write RNA on the tube to indicate it will be used in the RNA extraction.
  • Set up and label a yellow DNA (Spin-Away™) column in a collection tube for each sample.
  • Set up a waste beaker for liquid Zymo Kit waste. This will be disposed of in the waste disposal area in the collection container for Zymo Kit Liquid Waste.
  1. After the digestion, Invert/flick to mix tubes and centrifuge at 9,000 rcf for 3 minutes to pellet. Spin tubes with hinges facing out so pellet will form along the wall with the hinge.
    • Note on centrifugation: Depending on the centrifuge settings, it will count down time from when you press start or from when it reaches the desired speed. Ours counts down when you press start, so we add 30s to each spin as written to account for the time to get up to speed. Here, I would spin for 3 mins, 30s.
  2. Transfer all supernatant to a new, clean 1.5 mL microcentrifuge tube. Be careful to not disturb pellet.
    • Add: 345 uL (should be a 1:1 ratio with supernatant volume) of DNA/RNA Lysis Buffer
    • Mix these two solutions by pipetting up and down inside the tube.
    • Transfer the entire volume (~690 uL) onto the labeled Spin-Away™ filter column (will trap DNA, yellow colored) in a collection tube. Switch tips for each sample.
  3. Centrifuge at 15,000 rcf for 30 seconds and SAVE FLOW THROUGH from this spin.
    • Transfer the entire flow through volume (~690 uL) to the second labeled (RNA) 1.5 mL microcentrifuge tube.

DNA Extraction

  • Label your final DNA tube (clean 1.5 mL microcentrifuge tube) with the full sample number, “gDNA”, your initials, extraction date, and any other important information (like project, extraction specifications, etc.) on the side. The lid should say at least the full sample number and “gDNA”.
  • Label PCR tubes (we usually use PCR strip tubes) for a small aliquot of your sample for QC. Make sure your tube says the sample number (or a shortened version) and “DNA”.
  1. Transfer the yellow DNA column to a new collection tube.
    • Add: 400 uL of DNA/RNA Prep Buffer
    • Centrifuge at 15,000 rcf for 30 seconds and discard flow through in waste beaker (every time: do not tap tube on side of waste beaker to minimize splash-back).
  2. Add: 700 uL of DNA/RNA Wash Buffer
    • Centrifuge at 15,000 rcf for 30 seconds and discard flow through in waste beaker.
  3. Same as above: Add: 700 uL of DNA/RNA Wash Buffer
    • Centrifuge at 15,000 rcf for 30 seconds and discard flow through in waste beaker.
  4. Dry filter by centrifuging at 15,000 rcf for 2 minutes.
  5. Discard collection tube and transfer the yellow DNA column to the final, labeled DNA tube.
    • Add: 50 uL of warmed Tris buffer gently, directly onto the filter without touching the filter. (Elution)
    • Incubate for 5 minutes at room temperature.
    • Centrifuge at 15,000 rcf for 30 seconds.
  6. Repeat the elution step above for a total final volume of 100 uL of gDNA in the final tube. Discard yellow column.
  7. Aliquot 10 uL of your DNA into your labeled PCR tube for QC. Keep on ice.
  8. Place final DNA tubes in freezer box in -20 ºC.
    • Heating block can be turned off.

RNA Extraction

  • Set up and label a green RNA (Zymo-Spin™ IIICG) column in a collection tube for each sample.
  • Label your final RNA tube (clean 1.5 mL microcentrifuge tube) with the full sample number, “RNA”, your initials, extraction date, and any other important information (like project, extraction specifications, etc.) on the side. The lid should say at least the full sample number and “RNA”.
  • Label PCR tubes (we usually use PCR strip tubes) for a small aliquot of your sample for QC. Make sure your tube says the sample number (or a shortened version) and “RNA”.

Can do concurrently with DNA Extraction, the steps are the same after DNA Extraction Step 8/RNA extraction Step 20

  1. To the “RNA” tube from step 7:
    • Add: 700 uL of 100% Ethanol (1:1 ratio)
    • Mix these two solutions by pipetting up and down inside the tube.
    • Transfer 700 uL of this mixture to the labeled filter column (will trap RNA, green colored) in a collection tube. Switch tips for each sample.
    • Centrifuge at 15,000 rcf for 30 seconds and discard flow through in waste beaker.
  2. Transfer the rest of the mixture from the RNA tube (700 uL) onto the column. Discard empty tube.
    • Centrifuge at 15,000 rcf for 30 seconds and discard flow through in waste beaker.
  3. Add: 400 uL of DNA/RNA Wash Buffer
    • Centrifuge at 15,000 rcf for 30 seconds and discard flow through in waste beaker.
  4. DNAse digestion
    • Make DNase I treatment master mix (in a clean 1.5mL microcentrifuge tube):
      • 75µl DNA Digestion buffer x # of samples
      • 5µl DNase I x # of samples
        • stored in aliquots at -20 ºC, thaw on ice, never vortex!
      • Ex: for 8 samples, mix 600 uL of DNA Digestion buffer with 40 uL of DNAse I in a clean tube and invert gently to mix.
    • Mix gently. Do not vortex this mix.
    • Add 80 uL of this master mix directly onto each filter, gently without touching the filter.
    • Incubate at room temperature for 15 minutes.
  5. Add: 400 uL of DNA/RNA Prep Buffer
    • Centrifuge at 15,000 rcf for 30 seconds and discard flow through in waste beaker.
  6. Add: 700 uL of DNA/RNA Wash Buffer
    • Centrifuge at 15,000 rcf for 30 seconds and discard flow through in waste beaker.
  7. Same as above: Add: 700 uL of DNA/RNA Wash Buffer
    • Centrifuge at 15,000 rcf for 30 seconds and discard flow through in waste beaker.
  8. Dry filter by centrifuging at 15,000 rcf for 2 minutes.
  9. Discard collection tube and transfer the green RNA column to the final, labeled RNA tube.
    • Add: 50 uL of RNAse/DNAse free water (provided in kit) gently, directly onto the filter without touching the filter. (Elution)
    • Incubate for 2 minutes at room temperature.
    • Centrifuge at 15,000 rcf for 30 seconds.
  10. Repeat the elution step above for a total final volume of 100 uL of RNA in the final tube. Discard green column.
  11. Aliquot 10 uL of your RNA into your labeled PCR tube for QC. Keep on ice.
  12. Place final RNA tubes in freezer box in -80 ºC.

Quality Control (QC)

These steps analyze the quantity and quality of the DNA/RNA extracted.

RNA/DNA Quantity

Follow Broad Range dsDNA and RNA Qubit protocol to analyze sample quantity. Read standards once and record values, read all samples twice.

DNA Quality

If DNA quantity is sufficient (typically >10 ng/µL) follow the PPP Agarose Gel Protocol to determine DNA quality. “Good” DNA should form a distinct band a the very top of the gel.

Gel protocol for both DNA and RNA Quality (From Kristina Terpis)

  • Modified from this protocol
  • Set up small gel box and insert gel tray into the box with rubber gaskets touching the long side of the gel box. Make sure gaskets form a flush seal.
  • Make a thin 1.5% gel:
    • Add 0.75g of agarose and 50ml of 1x TAE (“New” container) to flask
    • Microwave for 45 seconds and remove from microwave using heat-protective glove.
  • Once cool enough to hold from the bottom of the flask, add 1 uL of gel green stain.
  • Swirl and pour into gel mold with two 14-well combs.
  • Once solidified (~30 minutes), cover with 1X TAE (“Used” container) as a running buffer
  • To each of the samples in the QC strip tubes (after Qubiting you should have about ~9 uL left),
    • Add 1uL of purple Tri-Track loading dye into each PCR tube, spin down to mix.
  • Load 3 uL of the 1kb GeneRuler ladder into the first well of each row. Load the DNA samples on the top row following the ladder and load the RNA samples on the bottom row.
  • Run the gel for 60 minutes at 60 volts.
  • Image using UV gel imager covered with black cover and orange filter.
  • Expectations
    • "”Good” DNA should form a distinct band a the very top of the gel.
    • “Good” RNA should form two distinct bands at the 18S and 28S locations about halfway down the lane.

RNA Quality

Proceed to this step if RNA does not appear clean on the gel. If RNA quantity is sufficient follow the Tape Station Protocol to determine RNA quality and obtain a RNA Integrity Number (RIN). “Good” RNA should have a RIN above 8.0 and form two distinct peaks at the 18S and 28S locations.

Written on October 3, 2022