DNA-RNA Extractions Porites astreoides homogenates, larvae, and adult fragments

Goal

To test the protocol for DNA/RNA extractions for airbrushed homogenates, larvae, and adult fragments of Porites astreoides for the Thermal Transplant 2017/2018 experiment.

Samples

I used 3 adult P. astreoides samples that were snap frozen, then airbrushed with filtered seawater. 0.5 mL of the homogenate was aliquoted then re-frozen at -80 °C. I also used 3 larval samples from 2018. 20 larvae were samples per vial. Additionally, I used 3 fragmented adult coral chips that were snap frozen, chipped, and re-frozen with 500uL of DNA/RNA shield and beads.

Sample Numbers

  • 17-215
    • Adult, R4-T2 (2017: Rim + Ambient Treatment)
    • Homogenate
  • 17-233
    • Adult, R11-T2 (2017: Rim + High Treatment)
    • Homogenate
  • 17-521
    • Adult, P19-T2 (2017: Patch + High Treatment)
    • Homogenate
  • 17-530
    • Adult, P6-T2 (2017: Patch + Ambient Treatment)
    • Homogenate
  • L-534
    • Larvae, R11-A (2018: Rim + High Treatment + Patch Transplant)
  • L-939
    • Larvae, P12-A (2018: Patch + High Treatment + Patch Transplant)
  • L-1026
    • Larvae, P16-A (2018: Patch + High Treatment + Patch Transplant)
  • 18-31-C
    • Adult, P9-A (2018: Patch + Ambient Treatment + Patch Transplant)
    • Fragment
  • 18-43-C
    • R15-B (2018: Rim + High Treatment + Rim Transplant)
    • Fragment
  • 18-57-C
    • P19-A (2018: Patch + High Treatment + Patch Transplant)
    • Fragment

Protocol description

I used a modified version of this protocolfor homogenates, this protocol for larvae, and this protocol for fragments.

Homogenate preparation

  1. Immediately after taking sample out of the -80°C freezer, add 500μL of DNA/RNA shield
  2. Once fully thawed, add 100 μL of Protenase K digestion buffer (Derived from volume of homogenate and 300/30 ratio from Zymo Protocol) and 50 μL of Protenase K (Derived from volume of homogenate and 300/15 ratio from Zymo Protocol)
  3. Vortex and spin down.
  4. Incubate on Thermomixer at 56°C at 1500 rpm for 30 minutes
  5. Spin down for 30 seconds to pellet any debris
  6. Remove 1000 μL of the supernatant and transfer in 5 mL tubes. Avoid any debris.

Larvae preparation

  1. Immediately after taking sample out of the -80°C freezer, add 1000μL of DNA/RNA shield
  2. Once fully thawed, add 100 μL of Protenase K digestion buffer (Derived from volume of homogenate and 300/30 ratio from Zymo Protocol) and 50 μL of Protenase K (Derived from volume of homogenate and 300/15 ratio from Zymo Protocol)
  3. Vortex and spin down.
  4. Incubate on Thermomixer at 56°C at 1500 rpm for 90 minutes
  5. Spin down for 30 seconds to pellet any debris
  6. Remove 1000 μL of the supernatant and transfer in 5 mL tubes. Avoid any debris.

Fragment preparation

  1. Take samples out from -80 °C.
  2. Add 500 μl of RNA/DNA shield to make the total volume 1 mL
  3. These samples already have beads in them.
  4. Vortex for 2 minutes. Leave the settings on and on max power.
  5. Remove 700 μl of the supernatant from the bead tube and place in a new 1.5 microcentrifuge tube labeled on the side with the extraction sample number and today’s date. Label the cap of the microcentrifuge tube with the sample number.
  6. Add 70 μl of Proteinase K digestion buffer (10:1 ratio of sample:digestion buffer), and 35 μl of Proteinase K (2:1 ratio of digestion buffer:Proteinase K) to the 5 mL centrifuge tube.
  7. Incubate on Thermomixer at 56°C at 1500 rpm for 30 minutes
  8. Spin down for 30 seconds to pellet any debris
  9. Transfer 700 μl of the supernatant to a new, labelled 5 ml centrifuge tube.
  10. Vortex and spin down all tubes.

Zymo Duet RNA DNA Extractions

Modified from the Zymo protocol.

DNA Extraction

  1. Add equal volume of DNA lysis buffer as sample volume to the 5 mL tube with sample
    • Vortex to mix
  2. Transfer 700 μL into DNA filter column (yellow)
    • Centrifuge at 16,000 rcf for 30 seconds
    • Remove flow through liquid and transfer into a new 5 mL tube labeled for RNA
  3. Repeat steps 2 until all liquid is gone
  4. Add 400 μL of DNA/RNA prep buffer to column
    • Centrifuge at 16,000 rcf for 30 seconds
    • Remove the flow through and transfer to waste
  5. Add 700 μL of DNA/RNA wash buffer
    • Centrifuge at 16,000 rcf for 30 seconds
    • Remove the flow through and transfer to waste
  6. Add 400 μL of DNA/RNA wash buffer
    • Centrifuge at 16,000 rcf for 2 minutes
    • Remove the flow through and transfer to waste
  7. Removed column and place into a new sterile 1.5 mL tube
  8. Add 50 μL of 10 mM Tris HCL (warmed to 55°C) directly to filter
    • Incubate at room temperature for 15 minutes
    • Centrifuge at 16,000 rcf for 30 seconds
    • Keep flow through in tube
  9. Add another 50 μL of 10 mM Tris HCL (warmed to 55°C) directly to filter
    • Incubate at room temperature for 5 minutes
    • Centrifuge at 16,000 rcf for 30 seconds
    • Discard column and keep flow through in tube
  10. Aliquot 10 μL into PCR tubes for QC.
  11. Store remaining aliquot at -20°C

RNA Extraction

  1. Add equal volume of 100% Ethanol as sample volume to the 5 mL tube with sample
    • Vortex to mix
  2. Transfer 700 μL into RNA filter column (green)
    • Centrifuge at 16,000 rcf for 30 seconds
    • Remove flow through liquid and transfer into a new 5 mL tube labeled for Protein
  3. Repeat steps 12 until all liquid is gone
  4. Add 400 μL of DNA/RNA wash buffer to column
    • Centrifuge at 16,000 rcf for 30 seconds
    • Remove the flow through and transfer to waste

  5. Make DNase I master mix: [75μL DNA digestion buffer and 5μL DNase] X n (sample #)

  6. Add 80μL of DNase I master mix directly to the filter of each column. Incubate at room temperature for ~15 minutes
  7. Add 400 μL of DNA/RNA prep buffer to column
    • Centrifuge at 16,000 rcf for 30 seconds
    • Remove the flow through and transfer to waste
  8. Add 700 μL of DNA/RNA wash buffer
    • Centrifuge at 16,000 rcf for 30 seconds
    • Remove the flow through and transfer to waste
  9. Add 400 μL of DNA/RNA wash buffer
    • Centrifuge at 16,000 rcf for 2 minutes
    • Remove the flow through and transfer to waste
  10. Removed column and place into a new sterile 1.5 mL tube
  11. Add 50 μL of RNAase-free water (warmed to 55°C) directly to filter
    • Incubate at room temperature for 5 minutes
    • Centrifuge at 16,000 rcf for 30 seconds
    • Keep flow through in tube
    • Made the gel during this incubation
  12. Add another 50 μL of RNAase-free water (warmed to 55°C) directly to filter
    • Incubate at room temperature for 5 minutes
    • Centrifuge at 16,000 rcf for 30 seconds
    • Discard column and keep flow through in tube
    • Took out qubit standards
  13. Aliquot 5 μL in PCR strip tubes for QC.
  14. Store remaining aliquot at -80 °C

Clean-up

  1. Place tissue and liquid in the waste container labeled Zymo extraction waste.
  2. Wipe down RNA free area with RNase away and kimwipes.
  3. Throw away all tips and restock tip boxes if necessary.

Testing Quantity and Quality

Quantify Results

Qubit

To test RNA and DNA quantity: Qubit

10 samples + 2 standards + 1 error = 13

DNA Broad Range

199 µl Buffer x 13 = 2587 µl Buffer 1 µl Reagent x 13 = 13 µl Reagent

RNA Broad Range

199 µl Buffer x 13 = 2587 µl Buffer 1 µl Reagent x 13 = 13 µl Reagent

Sample Type Sample DNA (ng/uL) RNA (ng/uL)
  Standard 1 188.26 393.91
  Standard 2 19093.72 8966.36
Homogenate 17-215 27.4 15.8
Homogenate 17-233 5.72 LOW
Homogenate 17-521 11.6 LOW
Homogenate 17-330 28.0 LOW
Larvae L-534 11.4 26.8
Larvae L-939 11.3 26.6
Larvae L-1026 10.6 25.8
Fragment 18-31 4.48 LOW
Fragment 18-43 7.08 13.6
Fragment 18-57 29.0 36.4

Gel Electrophoresis

To test DNA quality: Gel Electrophoresis

  • Today I did a small gel of the above protocol and ran it at 80V.

Gel

TapeStation

To test RNA quality: TapeStation

Overall Summary: Overall

Sample #17-215: Sample#17-215

Sample #1L-534: Sample#L-534

Sample #L-939: Sample#L-939

Sample #L-1026: Sample#L-1026

Sample #18-43: Sample#18-43

Sample #18-57: Sample#18-57

Written on August 5, 2020