We isolated RNA from D. melanogaster brains and treated the samples with the TURBO DNA-free™ Kit (Invitrogen). Prior to the treatment, the samples had concentrations of 27 ng/µl and 37 ng/µl RNA and RINe values of 6.8 and 7.2 (image 01). Afterwards the samples had RNA concentrations of 8.3 ng/µl and 12.4 ng/µl, but the RINe values could not be determined, because there were no markers (image 02). To me this does not look like the smear you would expect, if the RNA was degraded. Has anyone seen something similar and/or knows how to overcome this problem?
Hi, from what I have been told, a decrease in RNA concentration does not indicate degradation necessarily, as you would still get a similar reading if your RNA was degraded. I think the change in concentration might simply be due to repeating the measurement, because I usually don’t get the same reading even if I measure it again a minute later. Hope this helps.
- Your RNA is not degraded
- It is perfectly fine to use
- About 10 years ago I had a position where I prepared 100's of RNA samples for amplification for interogation by Affymetrix chips to procure genomic sweeps
- As part of that pipe line I had to assess RNA quality as you have done and on occasions came across what you are seeing. I came to the conlcusion it was an artifact of kinetic injection onto the chip and not diminuition in the quality of the sample itself: If you were to repeat your analysis but with more RNA loaded onto the chip you might get a different result
- If your RNA was degraded which I have seen you would not got sharp bands: Rather a more a generalised smear down the lane
And keep in mind that Bioanalyzer is not the best RNA measuring option. Qubit will give you more reliable and consistent results.
1. I concur with Kiril. The many samples i talked about and assessed were quantified with nanodrop; but qubit is even better
2. I also discovered the Agilent is not accurate for quantification
3. In terms of optimal amounts of RNA which will minimise the sorts of artifacts you experienced anything from 200ng to 500ng works well; if you cannot spare that much NEVER drop below 50ng
4. And make sure these amounts are verified with qubit or nanodrop not bioanalyser
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