Sam’s Notebook: RNA Isolation and Quantification – C.bairdi Hemocyte Pellets in RNAlater Troubleshooting

After the failure to obtain RNA from any C.bairdi hemocytes pellets (out of 24 samples processed) on 20200117, I decided to isolate RNA from just a subset of that group to determine if I screwed something up last time or something. Also, I am testing two different preparations of the kit-supplied DNase I: one Kaitlyn prepped and a fresh preparation that I made. Admittedly, I’m not doing the “proper” testing by trying the different DNase preps on the same exact sample, but it’ll do. I just want to see if I get some RNA from these samples this time…

Isolated RNA from the following 4 hemolymph pellet samples:

  • 6128_112_9 (Kaitlyn DNase)
  • 6204_114_9 (Kaitlyn DNase)
  • 6141_123_9 (Sam DNase)
  • 6245_126_9 (Sam DNase)

Isolated RNA using the Quick DNA/RNA Microprep Kit (ZymoResearch; PDF) according to the manufacturer’s protocol for liquids/cells in RNAlater.

  • Used 35uL from each RNAlater/hemocyte slurry.
  • Mixed with equal volume of H2O (35uL).
  • Retained DNA on the Zymo-Spin IC-XM columns for isolation after RNA isolation.
  • Performed on-column DNase step.
  • RNA was eluted in 15uL H2O

RNA was quantified on the Roberts Lab Qubit 3.0 using the RNA High Sensitivity Assay (Invitrogen), using 2uL of each sample.