I have done some qPCR and WISH for the same pool of embryos at 4hpf but the results are diferent, specially for ventral genes, such as vox and vent. The area of expression is reduced by doing WISH but the qPCR reflects no change. Any ideas why this happened?
I agree with Christopher Ford. In human tissue, I had this same problem comparing the results between microarray and qPCR. The problem was the reference gene. I wrote a paper about reference genes. For zebrafish I have used ef1a (Taqman System).
I am working with Xenopus and your problem is very common. For sure the reference gene choice is very important. If it could be possible try to dissect the area of interest before the RNA extraction. Some time, if yuor genes are not very much expressed their mRNA resulted diluted in the mass of the mRNAs of all the embryo! So dissecting out only the area in wich they are expressed could enrich the level of your specific mRNA and clear differences could emerge in your pPCR. Good luck!
P.S. If you have a good number of embryos analyzed (100 correctly injected embryos) trust your ISH!
I agree with all. QPCR rely heavily on your reference gene and no housekeeping gene is bound to as stable as people realize. The problem is made worse for people working on non-syngenic species like Xenopus, because each individual will display a huge variation of their overall transcriptional levels (Xenopus eggs vary in size from female to female!). It should not be the case in Zebrafish but…I wouldn't bet on it. Plus, remember that whatever treatment you did in your embryos may also affect the level of housekeeping gene transcripts. Trust your in situs!
Thank you all for your answers. They are very helpful. At first, we suspected the problem would be the reference and we changed to ef1a instead of 18s. It's important to say that the genes expressed in the dorsal side of the embryo such as gsc and flh are upregulated in the in situs as well as the qPCR but the ventral genes (vox and vent, both antagonist of gsc) are not downregulated in the qPCR, just in the in situs. That's the confussion for us.
I would suggest you use a couple different housekeeping genes if you still want to do qPCR. A paper published in 2008 suggests that these gene changes during development or certain treatment. I believe this is the paper:
Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment
Amy T McCurley and Gloria V Callard*
Great advices, I will have it in mind next time I perform the qPCR. Thank you very much for taking the time to comment on this.
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