I am trying to separate and concentrate tRNAs from high molecular weight (HMW) RNAs on an Ion Exchange Q sepharose FF GE column. tRNAs should come down at around 500-600 mM NaCl, and they do. However, I have very low binding efficiency of my HMW RNAs, as I find 90 percent of them in the flow-through. Does anyone know why this is? To what I know, all RNAs should bind to a stron ion-exchange matrix such as Q sepharose. I used Trizol to isolate the RNA, can that be the problem, that the structure of the RNA is somehow bad? Should I try with acid phenol?
Hi there,
If tRNAs are not binding to the matrix there is something wrong with your starting condition. What is the composition of the buffer of your starting sample? The binding of RNA to anion exchanger doesn't actually depend on the structuration of the RNA as it is about charge interaction.
BTW you may try Nucleobond kit from Macherey Nagel which is very performant for RNA purification. For info see the link below:
http://www.mn-net.com/Products/DNAandRNApurification/RNA/NucleoBondRNADNA/tabid/1325/language/en-US/Default.aspx
You probably have something competing for the binding. What's in the adsorption buffer?
What may be happening is that under your conditions (high salt?), the large RNA molecules are being excluded from the resin because of their size. In other words, you are seeing a gel filtration effect. (The Sepharose resin without the ion exchange functionality is used for gel filtration.) Q-Sepharose FF has an exclusion limit for globular proteins of ~ 4,000 kDa.
Actually, I was really careful not to interfere with binding of the RNA to the column, and I used just a 20 mM TRIS, 10mM KCl, 1.5mM MgCl2, 0.5mM DTT as an adsorbtion buffer. Then I elute with the increasing NaCl concentrations in the same buffer. I see tRNAs eluting at 500-600, which is what I am looking for, however, I see a lot of RNA in the flow through, not binding to my Q column at all, but in particular HMW RNAs. Is this maybe due to TRIZOL extraction of the RNA, do I mess up the RNA structure?
Your binding buffer is definitely low salt but what about the actual composition of the RNA sample you apply to the column? Is it identical to the binding buffer or is it different?