Zymo-Duet-DNA-RNA-Extraction-Protocol

Zymo Duet DNA/RNA Extraction Protocol for Gametogenesis January 2022 samples

Zymo’s Quick-DNA/RNATM Miniprep Plus Kit kit was modified for our lab.

Based on Putnam Lab protocols written by: E. Chille, K. Wong, and E. Strand.

Goal

Process DNA-RNA extractions for the 12-month Gametogenesis time series project in Mo’orea, French Polynesia which investigates the roles of parental effects and epigenetic inheritance in acclimatization and adaptation. The goal of my proposed research is to characterize maternal provisioning, epigenetic inheritance, and cross-generational impacts of thermal stress on Acropora pulchra throughout gametogenesis and early development (i.e., embryo, larval development).

Samples Remaining

January 2022 through August 2022 samples were brought back with CITES permit 20220819 and stored at URI in the flammable fridge (B) and the -80°C freezer (J).

  • January 2022, 25 samples remaining (12 processed 20220823)
  • February 2022, 36 samples remaining
  • March 2022, 40 samples remaining
  • April 2022, 40 samples remaining
  • May 2022, 40 samples remaining
  • June 2022, 40 samples remaining
  • July 2022, 40 samples remaining
  • August 2022, 40 samples remaining
  • September 2022, 40 samples remaining
  • October 2022, 40 samples remaining
  • November 2022, 40 samples remaining
  • December 2022, 40 samples remaining

Process

  • Samples were collected and preserved in DNA-RNA shield in Mo’orea, French Polynesia following the Sample Collection Protocol. Initial samples had 1-2 mL of DNA RNA shield added.

Zymo Duet DNA/RNA Extraction Protocol

12 January 2022 samples processed 20220823

timepoint tube.number collection.date colony.ID uL taken
January 2022 418 20220115 ACR-418 300
January 2022 422 20220115 ACR-422 300
January 2022 428 20220115 ACR-428 300
January 2022 431 20220115 ACR-431 300
January 2022 432 20220115 ACR-432 300
January 2022 438 20220115 ACR-438 300
January 2022 457 20220115 ACR-457 300
January 2022 458 20220115 ACR-458 300
January 2022 459 20220115 ACR-459 300
January 2022 460 20220115 ACR-460 300
January 2022 464 20220115 ACR-464 300
January 2022 465 20220115 ACR-465 300

image

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.

Proteinase K Addition

  1. Place 300 uL of the DNA-RNA shield aliquot into a new 1.5 mL microcentrifuge tube for extractions.
  2. Add 30 µl of PK digestion buffer to each sample tube (1:10 ratio of PK Digestion Buffer:Sample).
  3. Add 15 µl Proteinase K to each sample tube (1:2 ratio of Proteinase K:PK Digestion Buffer).
  4. Vortex for 5-10 seconds and spin down.
  5. Set timer for 15 minutes and let samples sit. Start labeling other tubes and organizing on tube racks.
  6. After 15 minutes, spin the samples down for 3 minutes at 16k rcf.
  7. After samples are spun down, remove the liquid into new 1.5 mL tubes, be careful not to disturb the pellet.
  8. Proceed to extraction steps.

DNA Extraction

  1. Set up yellow DNA spin columns and collection tubes, label appropriately
  2. Warm elution liquids to 70 degrees °C (10mM Tris HCl pH. 8.0 and RNase free water)
  3. Add equal volume 1:1 (345 µl) DNA/RNA lysis buffer to each sample tube
  4. Spin samples down for 1 minute at 16k rcf.
  5. Add 700 µl (total volume) of sample gently to the yellow DNA spin column
  6. Centrifuge at 16,000 rcf (g) for 3 minutes
  7. Important Save the flow through from this step: transfer to a new 1.5mL tube labeled for RNA
  8. Add 400µl DNA/RNA Prep Buffer gently to the yellow DNA spin columns
  9. Centrifuge at 16,000 rcf (g) for 1 minute
  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 1 minute
  13. Discard flow through (Zymo kit waste)
  14. Add 400 µl DNA/RNA Wash Buffer gently to the yellow DNA spin columns
  15. Centrifuge at 16,000 rcf (g) for 2 minutes 30 seconds
  16. Discard flow through (Zymo kit waste)
  17. Transfer yellow columns to new 1.5mL microcentrifuge tubes
  18. Add 50 µl warmed 10 mM Tris HCl to each yellow DNA column by dripping slowly directly on the filer
  19. Incubate at room temp for 5 minutes
  20. Centrifuge at 16,000 rcf (g) for 1 minute
  21. Repeat steps 18-20 for a final elution volume of 100 µl
  22. Label tubes, store at -20 °C.

RNA Extraction

  1. Add equal volume (700 µl) 100% EtOH to the 1.5mL 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 1 minute
  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 1 minute
  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
  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 1 minute
  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 1 minute
  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 and 30 seconds
  23. Discard flow through (Zymo kit waste)
  24. Transfer green columns to new 1.5mL microcentrifuge tubes
  25. Add 50µl warmed DNase/RNase free water to each green RNA column by dripping slowly directly on the filer
  26. Incubate at room temp for 5 minutes
  27. Centrifuge at 16,000 rcf (g) for 1 minute
  28. Repeat steps 25-27 for a final elution volume of 100µl
  29. Label 1.5mL tubes on ice afterwards, and aliquot 5µl into PCR strip tubes to save for Qubit and Tape Station to avoid freeze-thaw of your stock sample
  30. Store all tubes in the -80 °C

Extraction Content Analysis

These steps analyze the quantity and quality of the DNA/RNA extracted and may be done on a separate day from the extraction.

DNA/RNA Quantity

Follow Broad Range dsDNA and RNA Qubit protocol to analyze sample ++quantity++. Read all samples twice.

DNA/RNA Quantity - 12 January 2022 Samples

tube.number colony.ID qubit.reading.1 qubit.reading.2 quibit.average sample.type timepoint
DNA_standard_1 NA 200.58 NA 200.58 DNA.STD NA
DNA_standard_2 NA 24151 NA 24151 DNA.STD NA
1 ACR-418 25.6 25.4 25.5 DNA JANUARY
2 ACR-422 24 23.4 23.7 DNA JANUARY
4 ACR-431 25.4 24.8 25.1 DNA JANUARY
3 ACR-428 19.8 19.3 19.55 DNA JANUARY
5 ACR-432 15.1 14.8 14.95 DNA JANUARY
6 ACR-438 25 24.4 24.7 DNA JANUARY
7 ACR-457 27.6 27 27.3 DNA JANUARY
8 ACR-458 29.8 29 29.4 DNA JANUARY
9 ACR-459 13.1 13 13.05 DNA JANUARY
10 ACR-460 15.6 15.1 15.35 DNA JANUARY
11 ACR-464 19.2 18.8 19 DNA JANUARY
12 ACR-465 14 13.5 13.75 DNA JANUARY
RNA_standard_1 NA 422.43 NA 422.43 RNA.STD NA
RNA_standard_2 NA 9892.36 NA 9892.36 RNA.STD NA
1 ACR-418 31.6 31.6 31.6 RNA JANUARY
2 ACR-422 21.6 22 21.8 RNA JANUARY
3 ACR-428 27.6 27.6 27.6 RNA JANUARY
4 ACR-431 NA NA NA RNA JANUARY
5 ACR-432 16.8 16.4 16.6 RNA JANUARY
6 ACR-438 15.2 14.4 14.8 RNA JANUARY
7 ACR-457 18.4 18.2 18.3 RNA JANUARY
8 ACR-458 15.2 15.2 15.2 RNA JANUARY
9 ACR-459 17.4 16.6 17 RNA JANUARY
10 ACR-460 20.4 20.6 20.5 RNA JANUARY
11 ACR-464 13.8 13.8 13.8 RNA JANUARY
12 ACR-465 13.2 13 13.1 RNA JANUARY

We noticed that the RNA quantity for sample 431 was not detected, this could mean that there was possible contamination during the extraction process, or prior issues when the sample was taken that may have caused this. This sample will need to re-extracted and troubleshooted.

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. See example below:

annotated-biomin-gel-batches-4-5.png

DNA Quality - 12 January 2022 Samples

image of gel

  • Good DNA quality with strong and distinct bands at the top of gel.

RNA Quality

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. See example below:

TS-biomin-Ext-Batch-5-26.png

RNA Quality - 12 January 2022 Samples

You can also run RNA quality with a gel, which was done for our samples.

image of gel

We noticed that the RNA quality for sample 431 was not detected, this could mean that there was possible contamination during the extraction process, or prior issues when the sample was taken that may have caused this. This sample will need to re-extracted and troubleshooted.

Written on August 23, 2022