How do I know if my in situ hybridization probes binds mRNA and not genomic locus?
Usually not a problem because there are only two copies of genomic DNA, unless you are looking at repeats, and there should be hundreds of copies of your mRNA. Also, mRNA is mostly cytoplasmic whereas your genomic DNA must be nuclear.
Thanks.
Well, I'm looking for an mRNA that was reported to be in the nuclei (maternally deposited transcript of zebrafish).
But is there any technical separation? or the locus could be labeled too?
Hello Nir Haritan
For such method I feel you should use two standard controls:
1. RNase pre-treatment (which will demonstrate that detection is dependent upon the presence of RNA).
2. Opposite-strand nucleic acid sequences as probes (which will demonstrate sequence-specific nucleic acid strand dependency).
Best.
The first one is impossible I guess because I perform whole mount embryo.
But the second one is very nice! thanks.
Also, if you gene contains introns, you can design your probes on the exon-exon junction?
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