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Test RNA Extraction from Sea Stars with Zymo Direct-zol Kit

Testing RNA Extraction and Yield from Sea Stars with the Zymo Direct-zol RNA Miniprep Plus Kit

Notes:

  • All steps of the extraction other than the Tissuelyser and the centrifugation were done in the hood
  • All tips used were filter tips
  • Gloves, lab coat, mask, and glasses were worn at all times
  • Spaces and plastic materials were wiped with 10% bleach and RNaseZap before use
  • Forceps were cleaned with 10% bleach, DI water, 70% Ethanol and RNaseZap between uses
  • Samples were cut on foil

Sample Prep

  1. Samples thawed and temporary tube numbers:
    • CPAB002 -> # 3
    • CHSB005 -> # 4
  2. Took out sample 1 and placed small chunk in an empty bead tube

1

  1. Immediately added 800ul of tri-reagent to the bead tube (# 2), following recommendations from the Zymo protocol for solid tissues
  2. Took out sample 2 and placed small chunk in another empty bead tube

2

  1. Immediately added 800ul of tri-reagent to the second bead tube (# 3)
  2. I noticed that the liquid (DNA/RNA Shield) in the first tube had a lot of tissue chunks in it and I wondered if the tissue was mostly in the liquid and not in the big piece (seemed mostly like ossicles) so I added 400ul of the liquid to another bead tube (# 5)

3

  1. Sample tube # 2 had completely clear liquid but I also made a bead tube with 400ul of it just to be consistent (# 6)

4

  1. Added 400ul of tri-reagent to each of the bead tubes with liquid, following the recommendations from the Zymo protocol for liquid samples (1:1 ratio)
  2. Balanced the Tissuelyser with 4 tubes in each side (some empty) and ran it at 30Hz for 2 minutes (combination of recommendations from the Zymo protocol: “high speed” for 30-60 seconds, and the tissuelyer RNA from tissue recommendation 4 minutes at 30Hz)
  3. Briefly spun down tubes in the minifuge to collect beads at the bottom
  4. Tubes 3, 5, and 6 were opaque at this point, and the Zymo protocol says opaque liquid means incomplete lysis, and to add more tri-reagent and homogenize again

5

  1. So I added 400ul more tri-reagent to those 3 tubes and used the tissuelyser again for 2 minutes at 30Hz
  2. Briefly spun down the tubes again and now only # 3 looked opaque. There wasn’t enough room in the tube for more tri-reagent so I transferred 300ul of the liquid in tube # 3 to a new 1.5mL tube (3B) and added what was left of the tri-reagent (300ul) each to tube # 3 and tube # 3B and vortexed those two tubes and spun them down
  3. Then I transferred all the liquid (carefully not sucking up beads) from each tube (3, 4, 5, 6 and 3B) into new 5mL tubes, measuring the volume of liquid
    • 3: 850ul
    • 4: 600ul
    • 5: 900ul
    • 6: 800ul
    • 3B: 700ul
  4. Added equal volume 100% ethanol to each 5mL tube and invert to mix
  5. Used the large centrifuge to spin down the tubes briefly, then I noticed that in all the tubes there was a white pellet at the bottom, probably the really fine beads! I centrifuged them at max speed for a minute to pellet any beads. Now all the liquid was clear in all of the tubes!
    Tube 3 after 1st spin, still foggy but a lot of beads present 6
    Even already clear tube 6 had beads 7

Extraction

  1. Without sucking the beads up at the bottom I added 700ul from each 5mL tube to their respective spin columns and centrifuged at 16,000rcf for 30 seconds and discarded the flowthrough in the tri-reagent waste after
  2. I repeated the spins until all the liquid from the samples had gone through their columns
  3. I added 400ul of RNA wash buffer to each column, centrifuged, and discarded the flow through in the proper waste
  4. Created the DNase I mix: 375ul DNA digestion buffer and 25ul DNase 1
  5. Added 80ul of the DNase I mix to each filter by dripping
  6. Incubated for 15 minutes at room temp
  7. Added 400ul of direct-zol pre-wash to each column, centrifuged, and discarded the flowthrough in the proper waste
  8. Repeated the last step
  9. Added 700ul of the RNA wash buffer to each column, centrifuged for 1 minute, and discarded the flow through in the proper waste
  10. Transferred columns to new labeled 1.5mL tubes
  11. Added 50ul of RNase-free water to each column by dripping and centrifuged for 30 seconds
  12. Repeated the last step
  13. Aliquoted 5ul for qubit and gel into strip tubes and stored the final tubes in the grey -80

Qubit

  • Followed the Qubit protocol for broad range RNA
  • Used the 0ng/ul and 100ng/ul standards
standard 1 standard 2 3 4 5 6 3B
387 RFU 8548 RFU 27.4ng/μl 10.6ng/μl 15.5ng/μl too low 17.2ng/ul

TapeStation

Results Link

Written on September 15, 2020