Hi all,

I'm trying to purify viral RNA from water samples precipitated with ferric chloride. We are putting a dsRNA and a ssRNA virus isolates in a volume of buffer optimal for the virions. Viruses flocculate and precipitate and are retained by filtration. So we resuspend these viruses from the filters with citrate buffer.

Then we process the resuspended viruses to get the nucleic acids.

Final samples are treated with Turbo DNase and this inactivated with the reagent that precipitates.

Viruses that were not iron flocculated but directly extracted have decent concentrations of both viruses.

Nevertheless we keep obtaining at least some degraded RNA.

We are obtaining quite decent amount of viral dsRNA but ssRNA is really degraded or absent. 

We are afraid that a good part of the degradation is due to base-hydrolysis of RNA because of the presence of divalent cations such as Fe2+ (reduced from FeCl3 by citrate). Lysis of viral capsids is the first step after resuspension and is performed by heating at 55C the resuspended viruses with EDTA, SDS and proteinase K. We reduced the temperature from 65C to 55C due to the risk of acceleration of base-hydrolysis of RNA since possibly divalent cations such as Fe2+ and Mg+. 

this could be the problem?

EDTA or sodium citrate release the cations at this temperature?

We tried to remove the buffer containing Fe2+ by washing both by filtration and Amicon ultracentrifugation. But there is less recovery using these ways.

Is there a possible better way to get rid of the ions?  (if this is the real issue)

Do you think that Mg and Fe ions can precipitate with the isopropanol precipitation of nucleic acids and ethanol washes and be present in the final RNA samples and produced base-hydrolysis of RNA?

Actually in some electropherograms (RNA Pico 6000) Bioanalyzer) samples you can see evidence that there is something in the samples degrading the low-weight marker RNA.

Thoughts?

Best