Designing of RNA probe for in situ hybridization - (Nov/30/2011 )
Hi everybody. I would like to ask about general tips for choosing a specific region in a gene of interest for obtaining a DIG RNA probe for in situ hybridization. Which is the optimal size, %GC, if it´s better to use 5´or 3´regions, which hyb temperature is the optimal for a start....
Thanks to all!!!
I have used large fragments and even the entire gene. Cloned the whole gene (or 1000+ bp fragments) into a plasmid with a T7 or SP6 promoter, made DIG-RNA from that and then hydrolyzed the RNA into 150bp fragments. The protocol I used is similar to this (scroll down to page 6):
If you are concerned about cross hybridization, do a blast search -- if you find long hits (50+bp) of very high homology (very few mismatches), you could clone segments of your gene that exclude those regions.
Hyb temperature is related to your hyb buffer. The protocol above is done at 50deg, which is common when substantial amounts of formamide are included in the buffer.