how to get rid of antisense RNA - in situ hybridization (Jul/08/2010 )
During my in situ hybridization experiments I noticed that the sense and antisense probes gave the same signal. I use a 30 base oligo and I already use very stringent conditions. I just read about the existence of antisense RNA which might bind the sense probe. Does anyone know whether you can get rid of this antisense RNA? Because, how else can you check whether your signal is specific enough???
I have exactly the same issue with my in situs at the moment, and have not found an answer. Can you make your probe longer?
Nope, I've tried, but then it overlaps with many other mRNA sequences when I do a Blast, so then my specificity goes down again... Pfff, difficult!!!!
Hm... yeah. It is very annoying. Instead of making a longer probe can you make two small probes that do not overlap your sequence and see if you get the same staining?
I could do that indeed! There's also a company (Biognostik) which gives you three probes for your sequence + three control probes (actin-poly d(T) and nonsense), but it's rather expensive... The way it's going however, I think I'll turn to them. But that still won't fix the problem of sense RNA no? It's still going to bind no?
They have nope, I've tried, but then it overlaps with many other RNA sequences when I do a Blast, that appears to be targeted to the mRNA by the 25-nucleotide antisense nucleotide how else can you check whether your signal is specific enough.
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