Hello,
I am seeking for a method for isolation of HIV RNA from plasma sample. I am doing NGS so the isolate has to be highly pure (without any human or bacterial DNA/RNA). I tried standard kits for viral isolation and they are not working properly. Has anybody worked with this type of sample, and if so has anybody tried to use Molysis on viruses (in plasma samples)? I found some interesting methods for purification of my isolate and Molysis is one of them but my concern is that it will lyse my virus along with human and bacterial cells.
Hi Maja,
First of all, you have to pass the serum samples through 0.45 and then 0.22uM syringe filters in order to remove the bacterial and human contamination and then try to concentrate the VLPs or Virus Like Particales from your samples .
Then a lysozyme treatment followed by DNase and RNase treatment to remove the extracellular DNA and RNA. Then do a 3-4 time 1XPBS wash to remove the DNase and RNases.
Finally, you have to extract RNA by a manual Trizol method.
I do follow the same protocol for meta Virome.
Hope will help.
Hi Abhijit,
thank you for your help, I will surely consider using lyzozime treatment and as concerning the rest of the protocol, I have decided to pass my sample thorugh o.22 uM pore fiters and use DNase,/RNase pretreatment. Do you think that I can use 0.22 and 0.45 uM centrifugal devices instead of syringe filters, or would you recommend to definitely use syringes?
Kind regards,
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