Hello,
I will be collecting animal tissue samples (ranging from 5 to 50 grams per sample) primarily for virus titration. What I usually do is first I collect all my samples and store them at -70 °C and then I thaw them, cut into small pieces, homogenise in PBS, centrifuge and store at -70 °C again.
Now I would also like to isolate viral RNA from the tissue homogenates. Would it work if the homogenate is prepared the same way as described above and then used immediately on QIAgen RNA isolation columns?
Dear Wojciech ,
the method that you follow will lead to extraction of host nucleic acids in large quantities, as compared to very small amount of viral RNA of your interest. this will hamper your downstream virus nucleic acid assays. If you want to enrich viral particles specifically from your tissues and to remove host nucleic acids, you have to follow the VIDISCA or the TUViD-VM protocol. you may refer to the following papers for details, and one of our reviews on the subject.
best wishes,
SD
Article Next-generation sequencing in clinical virology: Discovery o...
Article Protocol for Metagenomic Virus Detection in Clinical Specimens
Article Identification of a new coronavirus
Greetings,
Ultracentrifugation gradient (iodixanol, sucrose, etc), always comes handy when isolating viral particles. Once isolated, RNA extraction through std. protocols (TRIzol, columns, phenol, etc) should be a piece very easy. All you have to do to know the ultracentrifugation conditions, is finding the specific density of the particular virus you are working with.
Best regards.
Homogenize tissue and usually we do freeze and thaw very often about 3 to 4 times to lyse cells and out those viral RNA. Once you get those lysed cells with medium or pbs, you can try to concentrate RNA by using ethnol or isopropanol precipitation.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus