I'm running RNA nanochip R6K in the TapeStation 2200 instrument. The samples correspond to molluscs and crustaceans Total RNA.
The issue we have is an overlapping of 18S and 28S peaks, therefore the software can't assign them. When we run electrophoresis gels, two clears bands can be noticed but wider than the total RNA from fish or other species.
Do any of you have experience with these problems? Can you suggest something?
Hello,
we have used the Tapestation to estimate the concentration of polyA mRNA after purification (consequently lacking the 5, 18 and 28S bands) and got some real issues. The problem with the Tapestation is that it doesn't use any ladder but an internal "scale" in the analysis software. This software usually assigns the lowest RNA band to be 5S and the highest to be 28S, as far as I understood.
I would suggest to run a known RNA ladder (for example the one used with the BioAnalyzer from the same company which provides the TapeStation) together with you samples.
When we ran our samples in parallel in both machines, it appears that TapeStation estimation of quantities are quite accurate, but size is problematic if you don't have a proper control or ladder.
Hope that helps,
Fabrice.
Thank you Fabrice, do BioAnalyzer's ladders run on TapeStation??.
I will try your suggestion!
regards,
Another possibility is that if you are observing the broader than usual 18S and 28S peak than your sample may contains bacterial RNA which can give you 16S and 23S peaks. This usually happens when we run RNA samples for mixed populations in metagenomic studies. Agilent software allow you to pick only one type of RNA prokaryotic or eukaryotic and not designed differentiate between them.
Hello Gustavo,
Have you considered the "hidden break" rRNA effect that occurs both in bivalves and crustaceans? 28S is processed into two fragments that (with the low resolution given by standard gel electrophoresis) looks like only one thick band around 18S. I'm not sure what you observe on the TapeStation 2200, but I have run intact oyster RNA (isolated from live animals just before evaluating the quality) in the BioAnalyser and I couldn't obtain two bands at 2:1 as with vertebrate RNA. This effect is a main concern that makes difficult to assess the RNA quality for RNA-seq and also for deplete the rRNA by using magnetic beads. Maybe the buffer chemistry of TapeSystem is giving you these discrepancies with the standard electrophoresis in your case. Please keep me updated about your case.
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