RNA Isolation and Quantification - C.bairdi Hemolymph Pellets in RNAlater

TL;DR - Recovered absolutely no RNA from any sample! However, I did recover DNA from each sample.

Isolated RNA from the following 23 hemolymph pellet samples:

  • 6128_112_9
  • 6204_114_9
  • 6141_123_9
  • 6245_126_9
  • 6240_134_9
  • 6260_136_9
  • 6257_138_9
  • 6259_139_9
  • 6258_140_9
  • 6255_143_9
  • 6256_146_9
  • 6265_155_9
  • 6266_156_9
  • 6261_164_9
  • 6120_165_9
  • 6251_167_9
  • 6262_168_9
  • 6243_173_9
  • 6263_179_9
  • 6264_180_9
  • 6200_208_12
  • 6204_252_12
  • 6190_256_12

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.


Qubit results (Google Sheet):

Well, none of these samples appear to have any RNA in them! The last time I did this, I started with 70uL of each sample and had yields high enough that cutting the sample volume in half should still have yielded ample RNA. This makes me think I screwed something up, particularly since I obtained DNA from each of the samples. However, I’ve reviewed all the steps and don’t see anything obvious that I forgot/screwed up.

I could re-quantify these using a higher volume, but I think it’s pointless. Samples that are too low for quantification on the Qubit are <1ng/uL. So, even if I were to increase the quantification volume to 5uL (i.e. 2.5x the volume I used initially), at best, the concentrations only be 2.5ng/uL. And, the the remaining volume of sample would be ~5uL; yielding 7.5ng of RNA in total. As such, I’ve discarded the samples and will attempt to re-isolate RNA from them.