DNA RNA Extractions Thermal Transplant Porites Trial 1

DNA/RNA extractions for Porites Thermal Transplant Trial #1 - ADULTS

Goal

To stream line the protocol for DNA/RNA extractions for Porites astreoides for the Thermal Transplant 2018 experiment

Samples

To start, I am using 5 adult P. astreoides samples that were already chipped (~0.5mm diameter) and placed in 500 uL of DNA/RNA Shield.

Sample Numbers: 29, 25, 33, 49, 23

Protocol description

I used a modified version of this protocol written by E. Strand. I only did the soft homogenization protocol because I am only interested in extraction coral DNA/RNA.

Protocol Preparation

Reagents and supplies

  • RNAse Away
  • RNA/DNA shield
  • RNAse-free water
  • Proteinase K digestion buffer
  • Proteinase K
  • Isopropanol
  • 10mM Tris HCl pH 8.0 made with RNase-free water
  • 10% bleach
  • Zymo Duet DNA/RNA Extraction kit buffers: DNA/RNA lysis buffer, DNA/RNA Prep Buffer, DNA/RNA Wash Buffer, DNA Digestion buffer, and DNase I

Equipment

  • Rocking oven that can be set at 55 °C
  • Thermomixer
  • Vortexer
  • Qiagen Tissue Lyser: Handbook
  • Bead tubes: 2mL 0.5mm glass beads and tubes from Fisher Scientific
  • Designated RNAse free space
  • Tabletop and larger centrifuges for 1.5 mL capable of 12,000xg
  • Clippers

Reagent 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.
  3. Reconstitute the lyophilized (freeze-dried) 20 mg Proteinase K with 1040 uL Proteinase K Storage Buffer or lyophilized (freeze-dried) 5 mg Proteinase K with 260 uL Proteinase K Storage Buffer. Vortex to dissolve. Store at -20 °C.

Soft Homogenization

  1. Thaw samples from -80 °C.
  2. Add 500 μl of RNA/DNA shield to make the total volume 1 mL
  3. Add ~0.25 μl of 0.5mm glass beads to each sample
  4. Vortex for 2 minutes. Leave the settings on and on max power.
  5. Remove 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. This supernatant will become the sample for “soft homogenization”.
  6. Add 300 μl of sample, 30 μl of Proteinase K digestion buffer (10:1 ratio of sample:digestion buffer), and 15 μl of Proteinase K (2:1 ratio of digestion buffer:Proteinase K) to a new 1.5 mL microcentrifuge tube.
  7. Vortex and spin down all tubes.
  8. Place bead tubes and leftover supernatant tubes (~300 μl) in -80 °C freezer.

Zymo Duet RNA DNA Extractions

Modified from the Zymo protocol.

DNA Extraction

  1. Set up yellow DNA spin columns and collection tubes, label appropriately
  2. Warm elution liquids to 70 °C (10mM Tris HCl pH. 8.0 and RNase free water)
  3. Add equal volume (to supernatant; 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. Important Save the flow through from this step: transfer to a new 1.5mL tube labeled for RNA
  8. Add 400 µl DNA/RNA Prep Buffer gently to the yellow DNA spin columns
  9. Centrifuge at 16,000 rcf (g) for 30 seconds
  10. Discard flow through (Zymo kit waste)
  11. Add 700 µl DNA/RNA Wash Buffer gently to the yellow DNA spin columns
  12. Centrifuge at 16,000 rcf (g) for 30 seconds
  13. Discard flow through (Zymo kit waste)
  14. Add 400 µl DNA/RNA Wash Buffer genetly to the yellow DNA spin columns
  15. Centrifuge at 16,000 rcf (g) for 2 minutes
  16. Discard flow through (Zymo kit waste)
  17. Transfer yellow columns to new 1.5 mL microcentrifuge tubes (“E1”)
  18. Add 50 µl warmed 10mM Tris HCl to each yellow DNA column by dripping slowly directly on the filter
  19. Incubate at room temperature for 5 minutes. Centrifuge at 16,000 rcf (g) for 30 seconds keep the flow through
  20. Add another 50 µl warmed 10mM Tris HCl to each yellow DNA column by dripping slowly directly on the filter
  21. Incubate at room temperature for 15 minutes.
  22. Centrifuge at 16,000 rcf (g) for 30 seconds
  23. Throw away filter and keep flow through.
  24. Aliquot 10 µl of the elution to 0.5 mL PCR tubes for Qubit and Gel Electrophoresis analysis.
  25. Store at 4 °C if quantifying the same day or the next, if waiting longer store in -20 °C freezer.

RNA Extraction Can do concurrently with DNA Extraction after DNA Extraction Step 7

  1. Add equal volume (700 µl) 100% EtOH to the 1.5 mL tubes labeled for RNA containing the original yellow column flow through
  2. Vortex and spin down to mix
  3. Add 700 µl of that liquid to the green RNA spin columns
  4. Centrifuge at 16,000 rcf (g) for 30 seconds
  5. Discard flow through (Zymo kit waste)
  6. Add 700 µl to the green RNA spin columns (the rest from the 1.5mL RNA tubes)
  7. Centrifuge at 16,000 rcf (g) for 30 seconds
    • Get DNase I from freezer
  8. Discard flow through (Zymo kit waste)
  9. Add 400 µl DNA/RNA Wash Buffer gently to each green RNA column
  10. Centrifuge at 16,000 rcf (g) for 30 seconds
  11. Discard flow through (Zymo kit waste)
  12. Make DNase I treatment master mix:
    • 75µl DNA Digestion buffer x # of samples
    • 5µl DNase I x # of samples

      Today I had 5 samples, therefore: 375 µl of DNA Digestion buffer and 25 µl of DNase I

  13. Add 80 µl DNase I treatment master mix directly to the filter of the green RNA columns
  14. Incubate at room temp for 15 minutes
  15. Add 400 µl DNA/RNA Prep Buffer gently to each column
  16. Centrifuge at 16,000 rcf (g) for 30 seconds
  17. Discard flow through (Zymo kit waste)
  18. Add 700 µl DNA/RNA Wash Buffer gently to the green RNA spin columns
  19. Centrifuge at 16,000 rcf (g) for 30 seconds
  20. Discard flow through (Zymo kit waste)
  21. Add 400 µl DNA/RNA Wash Buffer genetly to the green RNA spin columns
  22. Centrifuge at 16,000 rcf (g) for 2 minutes
  23. Discard flow through (Zymo kit waste)
  24. Transfer green columns to new 1.5 mL microcentrifuge tubes
  25. Add 50 µl warmed DNase/RNase free water to each green RNA column by dripping slowly directly on the filter
  26. Incubate at room temp for 5 minutes
  27. Centrifuge at 16,000 rcf (g) for 30 seconds
  28. Repeat steps 25-27 for a final elution volume of 100 µl
  29. Aliquot 5 µl of the final elution to 0.5 mL PCR tubes for Qubit and TapeStation analysis.
  30. Store all tubes in the -80 °C freezer.

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

To test RNA and DNA quantity: Qubit
To test RNA quality: TapeStation
To test DNA quality: Gel Electrophoresis

Solution Key:

DNA/RNA Lysis Buffer: Contains a high concentration of chaotropic salts, which destabilize hydrogen bonds, van der Waals forces and hydrophobic interactions. This destabilizes proteins, including nucleases. The buffer will also disrupt association of nucleic acids with water.
DNA/RNA Wash Buffer: Used to remove residual proteins and carbohydrates, and purifies the molecular end product.
DNase I: deoxyribonuclease is an enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone, thus degrading DNA. This is used in the RNA extraction step to purify RNA final product.
DNA/RNA Prep Buffer: Ethanol and guanidinium chloride used a deproteinization measure. Guanidinium is a very strong denaturing agent that will dissolve cells and RNAses.
EtOH: the addition of alcohol further enhances and influences the binding of nucleic aicds to the silica (see spin colum description below).
10 mM Tris HCl: DNA is more stable at slightly basic pH and will dissolve faster in a buffer than water. Using Tris instead of water will enhance rehydration of high molecular weight DNA.
DNA/RNase free water: Used as an elution in the RNA extraction steps. RNA can tolerate a slightly acidic pH and dissolves. Warmed water as an elution increases high molecular yield.
Yellow DNA/Green RNA Spin Columns: contain a silica membrane that binds to nucleic acids. Proteins and polysaccharides should be in the flow-through liquid that is discarded while RNA and DNA is kept on the membrane.
Proteinase K: an enzyme that cleaves the peptide bond in proteins next to the carboxyl group of hydrophobic amino acid residues. This decontaminates the sample and leaves purified DNA/RNA.

General workflow of an extraction:

  1. Cell lysis through disruption of cellular membranes
  2. Dehydration and precipitation of the cellular proteins (protein denaturation)
  3. Separation of cellular proteins and other cellular components out of the nucleic acid
  4. Precipitation and dissolving the nucleic acid

Quantify Results

Qubit

5 samples + 2 standards + 1 error = 8

DNA Broad Range

199 µl Buffer x 8 = 1592 µl Buffer 1 µl Reagent x 8 = 8 µl Reagent

RNA Broad Range

199 µl Buffer x 8 = 1592 µl Buffer 1 µl Reagent x 8 = 8 µl Reagent

  • The first row indicates the DNA qubit results using the DNA broad range standards with black writing on the cap. The standards were very low, therefore I repeated the qubit results with the DNA broad range standards with red writing on the cap.
  S1 S2 23 33 25 49 29
DNA (Standard with black writing on cap) 104.98 9261.30 3.18 2.8 Low Low 5.18
DNA (Standard with red writing on cap) 162.7 21982.36 3.9 3.08 2.18 2.08 5.38
RNA 382.72 10541.49 15.6 12.2 13.4 11.6 25.8

TapeStation

  • I did not tape sample 49 because there were the open tape only had 5 spots (4 samples + ladder). I will tape this sample at a future date if necessary

Overall Summary: Overall

Sample #29: Sample#29

Sample #33: Sample#33

Sample #23: Sample#29

Sample #25: Sample#25

Summary of results

  • Overall, there were very low concentrations of DNA and RNA, however the quality of the RNA is relatively good for 2 samples (Sample 23 and 29).
  • I did not do a heating digestion step according to this modified protocol from E. Strand. P. astreoides has lots of mucus, therefore a heating digestion step with the Proteinase K might be necessary

Next steps

  • Tomorrow I will try a heating step with the remainder of the bead-bashed supernatant to determine if this will increase the yield of DNA and RNA.
Written on December 3, 2019