Hi, in our lab we have been using Dynabeads mRNA direct to extract mRNA from beetles. According to the manufacturer's protocol, re-using the beads after the first elution should minimize rRNA contamination. However, what we have been finding out through bioanalyzer is that doing one or two elutions makes no difference for the contamination found in the extracted mRNA. We always get ~25% rRNA contamination, no matter what. That is usually only one peak, which seems to be 28S based on the size. I am attaching a figure with typical results.
Has anyone had any similar experiences and found a work around?
Hi Bruno. On general when you purify mRNA using different kits you always have some residual rRNA, the point is to have as little contamination as possible. Currently I am using PolyATtract® mRNA Isolation Systems and I am quite satisfied.Good luck.
Hey Yordan,
Thanks for your answer. I expected some residual rRNA, but I was surprised by the behavior of the kit. Doing two elusions consumes more time and reagents, and it does not seem to be doing much. In any case, we will soon prepare libraries from our extracted RNA and see how they turn out.
Hi,
Alternative/ additional ways of getting rid of rRNA contamination's:
(1) increase the temperature of the washing step to 37oC
(2) lower the salt concentration in the washing solution
(3) wear gloves, work on ice
(4) increase volume of washing buffer used
(5) increase the number of washing steps
kind regards
ketil
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