I was wondering if anyone could advise me on RNA quality.
I used a Qiagen RNeasy kit to isolate RNA from yeast and an Agilent Bioanalyzer to check the quality of the RNA (see attached). Yeast cultures were treated with either water of formaldehyde (final concentration 600 uM), and cells were harvested 30-60 minutes after treatment.
According to both a Nanodrop reading and the Bioanalyzer, my yields were ~600-1000ng/ul, This is more than enough RNA to make a library with, provided it is good RNA. The A260/280s were ~2.1, indicating minimal protein contamination, and the A260/230s were ~2.0-3.0, suggesting little or no guanidinium or phenol contamination.
In spite of this, I am concerned about the quality of my RNA. There are definitely strong 18S and 25S rRNA peaks, but they are less pronounced than in pictures I have seen online. The automatically calculated RNA Integrity Score, or RIN, is ~4 for samples without formaldehyde and ~2.5 for samples that were treated with formaldehyde. This is doubly disconcerting since the purpose of my study is to ascertain rapid transcriptional changes concomitant with formaldehyde stress--and one of the aspects of formaldehyde stress is the formation nucleic acid adducts.
The first set of RNA samples were collected by someone else. To eliminate the possibility of user error, I performed the extractions myself and bought a new Qiagen kit before doing so, in case the kit components were contaminated. My RNA yields were about 20 times higher than the previous person's had been, but the overall RIN scores and shapes of the electropherograms were strikingly similar.
Based on the pictures I have attached, what do you think? Does my RNA look degraded? If so, do you think that the RNA extraction or cell collection procedure itself could be to blame? To lyse my yeast, I treat previously flash-frozen cells with Zymolyase for 30 minutes at 30 degrees C. The zymolyase works great at breaking open the cells, but I wonder if the treatment of cells at 30 C for 30 minutes is long enough for them to start acting up and destroying their RNA. It would seem like this is unlikely, since if it were, the product would not be that popular. Then again, maybe no one has reported this yet since no one has tried to perform RNAseq following zymolyase treatment before (or if they have, they have chosen not to talk about it). Do you think I would have better luck trying a method with phenol and chloroform?
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