Tuesday, October 31, 2006
Problems with the riboswitch assay
This week I got the preliminary riboswitch experiment results. I tested sxy RNA for binding ATP, GTP, dATP. dGTP, guanosine, adenosine, AMP and GMP (separately). Each of these substances was incubated in three different concentrations (10 nM, 300nM and 100uM) with labeled sxy RNA in a special buffer that contains Mg and K. I also had three controls:
1) RNA incubated with 100uM of ligand and not digested with RNase (negative control)
2) no_ligand control, sxy RNA with no ligand added and digested with RNase
3) ladder control, which is sxy RNA digested with RNase under denaturing conditions so that it cuts all of the C and U residues in the sequence (not only single stranded ones).
The results did not seem very exciting at the first glance, since all of the samples gave the same result. Further investigation revealed the assay is actually not working properly. First, all of the samples gave the exact same bandage pattern and the same intensities of the bands. This would be fine and I would have concluded that the ligand addition is not affecting folding of sxy RNA, if only wasn't for my controls. All of my negative controls were digested in the exact same way as the rest of the samples (these RNAs should be intact since no enzyme was added). The very first thing I suspected was RNase contamination. However, if this has really happened we would expect to see more cleavage in the samples where I added RNase (which is not the case).
Another observation was that all of the samples except for the ladder control had very high background, meaning that there is lot of unspecific cleavage (that gives fragments of various lengths and resolves as darker background in a gel).
One of the explanations of what happened is that the buffer that I used for riboswitch analysis was somehow affecting RNase activity or (and) unspecifically digesting the RNA. The unspecific digestion of RNA usually happens when the solvent is too basic. I checked the pH, concentration and composition of the riboswitch buffer and it is not much different in composition from the regular buffer (the one that I use for secondary structure probing). The only thing that is different is pH - the riboswitch buffer is more basic (pH 7.9) than the regular one (pH 7). According to the technical support that I called, this shouldn’t make much of a difference for RNase activity however, I suspect that this is the major problem in the assay. I did bunch of controls yesterday testing all of the buffers and reagents that I use in the experiment and ran the samples in a gel. I should have the answer of what is going on later today. If it turns out that the riboswitch buffer is the one to blame, I think I will try doing the assay using the regular buffer for riboswitch analysis.
Another news is that I got my thesis back form all of my committee members and I am currently working on the revisions. This shouldn’t take too long and I think that I‘ll have everything done and ready to go by the beginning of next week.
1) RNA incubated with 100uM of ligand and not digested with RNase (negative control)
2) no_ligand control, sxy RNA with no ligand added and digested with RNase
3) ladder control, which is sxy RNA digested with RNase under denaturing conditions so that it cuts all of the C and U residues in the sequence (not only single stranded ones).
The results did not seem very exciting at the first glance, since all of the samples gave the same result. Further investigation revealed the assay is actually not working properly. First, all of the samples gave the exact same bandage pattern and the same intensities of the bands. This would be fine and I would have concluded that the ligand addition is not affecting folding of sxy RNA, if only wasn't for my controls. All of my negative controls were digested in the exact same way as the rest of the samples (these RNAs should be intact since no enzyme was added). The very first thing I suspected was RNase contamination. However, if this has really happened we would expect to see more cleavage in the samples where I added RNase (which is not the case).
Another observation was that all of the samples except for the ladder control had very high background, meaning that there is lot of unspecific cleavage (that gives fragments of various lengths and resolves as darker background in a gel).
One of the explanations of what happened is that the buffer that I used for riboswitch analysis was somehow affecting RNase activity or (and) unspecifically digesting the RNA. The unspecific digestion of RNA usually happens when the solvent is too basic. I checked the pH, concentration and composition of the riboswitch buffer and it is not much different in composition from the regular buffer (the one that I use for secondary structure probing). The only thing that is different is pH - the riboswitch buffer is more basic (pH 7.9) than the regular one (pH 7). According to the technical support that I called, this shouldn’t make much of a difference for RNase activity however, I suspect that this is the major problem in the assay. I did bunch of controls yesterday testing all of the buffers and reagents that I use in the experiment and ran the samples in a gel. I should have the answer of what is going on later today. If it turns out that the riboswitch buffer is the one to blame, I think I will try doing the assay using the regular buffer for riboswitch analysis.
Another news is that I got my thesis back form all of my committee members and I am currently working on the revisions. This shouldn’t take too long and I think that I‘ll have everything done and ready to go by the beginning of next week.
# posted by Okapia Johnstoni @ 5:05 PM 
