Isolating RNA from 2018-19 hemolymph samples

I decided to use the Qiagen RNeasy micro prep kit because the max capacity is 45ug and you can elute in as little as 10uL. The Zymo microprep DNA/RNA kit columns have a max binding capacity of 5ug DNA and 10ug RNA. One concern I had not knowing how many cells are in these sample was overloading the column which can lead to low yield, impure RNA, and inefficient isolation.

To test the protocol out, I used a couple of old samples that are not part of the experiment. These were hemolymph samples from Nov. 27 2018 from two adults that I reared at UW.

Prep work: Made 80% ethanol (4mL RNase free H2O + 1mL 100% EtOH) and 70% ethanol (3.5mL RNase free h2O + 1.5mL 100% EtOH).

I followed the standard protocol as follows:

  • Added 350uL Buffer RLT plus to pellets, vortexed
    • one sample (“star”) has visible tissue in the pellet that didn’t full dissolve
  • Pipetted lysate onto QIAshredder spin column and spun at max speed for 2 min.
  • Transfered homogenized lysate to a gDNA eliminator spin column and spun at max speed for 30 sec.
  • I reserved 175uL of the homogenized lysate after gDNA elimination in an eppy and labeled it as “Chewy 11/27 RNeasy HM” and “Star 11/27 RNeasy HM” and saved in -80C in Rack 5, Row 1, column 3. This was in case I screwed up on the prep that I’d have something to go back to.
  • Added 200uL of 70% EtOH to flow through, pipetted up and down to mix, then transfered to an RNeasy MinElute spin column and spun at max speed for 15 sec.
  • Added 700uL Buffer RW1 to the column and spun at max speed for 15 sec, dicarded flow through
  • Added 500uL RPE buffer and spun at max speed for 15 sec, then discareded the flow through. I repeated this step by accident
  • Added 500uL of 80% EtOH and spun for 2 min at max speed
  • Transferred mini column to clean collection tube and spun for 3 minutes on max speed to dry column. Then remembered to open the lid and spun again for an additional 4 minutes
  • Transferred column to a clean 1.7mL eppy, added 15uL RNase free H2O and spun at max speed for 1 min to elute. Pipetted the residual liquid on the edge of the column directly onto the column and spun again for 1 min.

Quantifying RNA


  • I measured 1 uL of RNA with the Qubit HS RNA kit.
    • Star: too low for detection
    • Chewy: 38.0 ng/uL
    • Standard 2: 10.2ng/uL


Plan for tomorrow

Hemocyte samples to prep:

  • location: Rack 6, col.2, row 2:

    1. 053
    2. 060
    3. 015
    4. 7
    5. 025
    6. 057
    7. 3
    8. 6
    9. 8
    10. 14
  • location: Rack 5, col.3, row 1

    1. 021
    2. 022
    3. 023
    4. 024
  • Prepare enough reagents for 20 preps:
    • Add 350uL RLT/sample = 7mL RLT + 70uL B-ME (-20C 2nd shelf drawer 7)
      • this will help with dissociated proteinaceous tissue and help denature RNases
  • Label all tubes:
    • qiashredders (14)
    • gDNA eliminator columns (14)
    • minElute columns (14)
    • eppys (2 x 14)
    • qubit (16)