I synthesized a specific fluorophore probe. It can bind with a specific site on DNA, but the problem is maybe it can also bind with RNA. So I need to show that this probe results in only DNA, not RNA. Is there any assay that I can use to determine the probe only specifically bind on DNA? I performed 2 experiments
1. EMSA shown that the probe specific binds to DNA
2. This experiment followed the first one, I perform fluorescent binding assay. Here, I cannot show that the probe specific bind with only DNA.
Thank you and hope to receive your responds soon.
If the sequence is common to both DNA and RNA it will bind to both. If you can, try designing your probe in an intron of your DNA. That way you won't find it in the RNA and you won't need to do any experiment.
You can treat your samples with RNAse to remove all RNA, with RNAse-free DNAse to remove all DNA (negative control) or (if, as i presume, your probe is DNA) specifically remove probe-bound RNA only with RNAse H
I really approriate your help. I am performing my experience with RNAse. Hope good results.
You can do the EMSA experiments with an RNA oligo. There are known RNA binders that you can use as positive controls.
Divide your sample into two identical tubes, then treat each one the samples with/without RNAase, then do the assy you want.
Regards / Beston
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