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Experimental Design Problem for RNA FISH


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#1 Katy

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Posted 15 September 2009 - 11:36 AM

Hello Everyone,

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.

Any thoughts?

Thanks!

#2 mdfenko

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Posted 24 September 2009 - 07:47 AM

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?


don't overlap the probes.
talent does what it can
genius does what it must
i do what i get paid to do




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