Experimental Design Problem for RNA FISH - (Sep/15/2009 )
I'm interested in detecting both ssRNA and dsRNA in my cells by FISH. The problem I'm encountering is that if I design probes against the (+)RNA, and then probes against the (-)RNA (labeled with different fluorochromes), how can I detect both probes in the same cell without having the probes hybridize to each other?
Is there such thing as sequential FISH? (ie. add the (+) probe, hybridize, wash, add (-) probe, hybridize wash, etc).
I can't use an antibody against dsRNA in combination with probe labeling because the antibody will also detect the probe/RNA combination and give a false positive.
Will I end up having to do three slides...one with the (+) probe, one with the (-) probe and one with the antibody against dsRNA.
Ideally, I would like to have everything detected in the same cell...much more informative that way.
Katy on Sep 15 2009, 03:36 PM said:
don't overlap the probes.