Test RNA extraction

I just noticed that the last time I posted was about a year ago – wow, time flies! I have primarily been working on RADseq data assemblies (i.e., being a computer jockey) and have not done really any bench work since then, hence the gap.

I’m finally getting into some RNA extractions on both coral and anemone samples. The coral samples are Porites astreoides from the Belize transplant experiment, collected in November 2016. The anemone samples are Anthopleura elegantissima from Natalie Coleman’s OA experiment last summer. Both sets of samples were flash frozen in liquid nitrogen and stored in a -80°C freezer since then. In January, I crushed the majority of these samples to a fine powder under liquid nitrogen using a mortar and pestle. The coral samples ended up being more substantial and I only needed a portion of each, and the crushed powder I obtained weighed approximately 500-700 mg per sample. The anemones samples, on the other hand, were smaller, and I only got about 60-150 mg per sample. Granted, some of the weight of the coral samples is probably skeleton.

For the extractions, I used the Qiagen RNeasy kit, with initial sample homogenization with Qiashredder columns. I used the animal tissues protocol with a final elution volume of 40 µl (single pass). The following are results from the Qubit RNA HS assay:

 

Organism Sample RNA (ng/ul) Sample vol (ul) Notes
P. astreoides Past13 ’16 Home 35.9 40
P. astreoides Past13 ’16 CBC 45.7 40
A. elegantissima H2 End 422 too low 40 Low amount of starting material; thawed slightly
A. elegantissima A2 End 6 too low 40 Low-ish starting amount
A. elegantissima M3 End 468 124 60 Had to be diluted to Qubit
A. elegantissima A1 end 425 71.6 60 Had to be diluted to Qubit

 

Overall I am pleased with these results. The only one I find odd and hard to explain is H2 End 422 – this had a relatively low amount of starting material, but I didn’t think it was that low. It may have thawed or been otherwise compromised, but I made no note of this.

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