In the continuing struggle to isolate RNA from the Chionoecetes bairdi hemolymph preserved in RNAlater, Pam managed to find the Quick-DNA-RNA Microprep Plus Kit (ZymoResearch) as a potential option. We received a free sample of the kit and so I gave it a shot. Grace pulled 10 samples she’d previously used to isolate RNA (and was unable to get anything out of virtually all of them using the RNeasy Plus Micro Kit (Qiagen)) for me to try out this new kit:
- 31
- 142
- 285
- 291
- 317
- 326
- 419
- 455
- 503
- 506
I’ve included an image of the sample tubes, due to the fact that they are colored, and I can’t remember the significance (insignificance?) of this. Want to make sure that is documented in case it’s important.
Used the same starting volume of hemolymph/RNAlater “slurry” as Grace: 15uL
Then followed the Zymo protocol for “Samples in RNAlater” (used H2O as intial sample diluent).
- I did not perform the DNase step. This should be performed for all future preparations, though.
Eluted with 15uL.
Quantified RNA using the Roberts Lab Qubit 3.0 and the RNA High Sensitivity Assay (Invitrogen).
Used 5uL of each sample.
The news is most excellent! There are detectable levels of RNA in all samples!
RESULTS
Qubit data (Google Sheet):
| SampleID | Concentration (ng/uL) |
|---|---|
| 31 | 6.24 |
| 142 | 9.2 |
| 285 | 9.92 |
| 291 | 3.49 |
| 317 | 10.1 |
| 326 | 11.6 |
| 419 | 8.28 |
| 455 | 7.2 |
| 503 | 7.64 |
| 506 | 15.6 |