I would like to check the expression of two housekeeping genes, which, based on RT-PCR results, are expressed throughout development (most likely in a ubiquitous manner). Yet I just can't make the in situs to work. I tried to clone different bits of the cDNAs for probe synthesis, and used different temperatures (60C/65C/70C) for hybridization, but there is no staining whatsoever. Any ideas...?
Hi Mate,
have you tried making the probe with cDNA from clones of the the 5' or 3' UTRs?
At least for one of them yes, now I'm using a fairly big part of the 3'UTR. (For the other gene the VEGA annotation is "3' incomplete", so there only a part of the CDS was cloned, and the 5'UTR is too short)
One possible explanation is that your gene is expressed very weak in a cell, but ubiquitously. What do you think?
Hi Shingo, yes that is a possibilty. Yet I do think at least for one of them, in fast proliferating tissues this cannot be the case (there is some ZFIN expression data on one of them from the Thisse screen).
Just to follow up on this: in the end there was nothing extraordinary going on. After changing all the solutions and reagents, things started to work. (As even before some probes gave nice signals, it was not obvious that this ought to be done, nevertheless the drastic solution solved the problem.)
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