I am trying to separate RNA binding sequences to virus particles from non-binders through sucrose centrifugation. I am layering the RNA virus particle mix onto 20% sucrose and taking the pellet out, which should have the virus particle with the bound RNA, whereas the supernatant should have the unbound RNA. I was wondering whether RNA ever enters the sucrose or if it continues to sit at the top and never gets into the sucrose layer. I would really like to clear my concept here, any thoughts would be really appreciated.
Thanks for the answer Shruti ! The experiment is a little different, I don't use a sucrose "gradient". I take my virus and RNA mix let it reach equilibrium and then layer it onto 20% sucrose. I centrifuge them and take the pellet assuming that the pellet will have the RNA bound to the virus whereas the supernatant will have only the unbound RNA. Where I am getting confused is, this is like simple centrifugation, so since virus is bigger in size it will sediment faster and pellet whereas RNA won't. But, I was wondering why to use sucrose in first place and secondly how can I be sure that the unbound is not coming along with the virus particle. From what I've read is that the density of RNA is more than sucrose so it should also pellet. Let me know your thoughts !
I guess your experiment is similar to a regular pull-down. Difference is using virus particles instead of resins. So the operation should be the same after you get the pellets, That is, you should wash them several times, collect your washes and final cleaned pellets and examine those materials to confirm the specific binding of your RNAs to the target.
You can include a control by using only your RNA without virus and see how much of free RNA you find in your bottom fraction (the pellet), in the sucrose layer, and on top of the sucrose cushion where you've applied the sample. Hope this helps. Good luck.
Thanks for your suggestion Dr.Ortmann . I was wondering as to why do we even add the sucrose layer. If we just centrifuge the mix, by the virtue of size of the particles shouldn't they separate ??
There are 2 kinds of centrifugation used to separate molecules: 1) equilibrium density gradient, in which the solution starts out uniform and a gradient is formed during the centrifugation and molecules are separated based on their densities (not size), and 2) sucrose gradient rate centrifugation, in which the sucrose serves as both a stabilizer of the tube contents (analogous to a gel in gel electrophoresis) to minimize mixing as fractions are collected (representing distances down the gradient/tube) and with the right gradient shape an isokinetic gradient can be formed so that molecules (e.g., DNA) can travel at constant rate and be separated by their size (same density). Most of this is well explained in Physical Chemistry books. Much of this literature was published in the 1970s and 80s. RNA id somewhat more difficult to size separate by rate centrifugation because it is more flexible than DNA (single vs double stranded).
In your case, seeking to separate RNA bound to a large object like a viral particle, the rate sedimentation should work fine; you just have to work out how long it takes for your virus to get to the bottom (or pellet) before sufficient unbound RNA (run separately as a control) gets there. Run separate tubes for (1) virus alone, (2) RNA alone, (3) your mixture and see how long a run gives optimal separation and purity.
I suggest that you switch to RNase free sucrose. Regular p.a. sucrose can be treated with charcoal to be essential RNase free.
I am answering this very late, but I don't know why nobody come with the CsCl ultracentrifugation. If centrifugued for enough time you should have virus, virus-RNA, and RNA all separated.
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