Puzzling Real Time Results - (Nov/19/2010 )
We have a very puzzling situation in our lab and hopefully someone can help us!
We have identified a point mutation that causes a truncated form of the resulting protein. We know that the mutation is homozygous lethal and zebra fish embryos will die after 7 days and the heterozygous form is viable. I have been performing qPCR using 5 day embryos, 1 year old adults and 3 year old adults. I have run real time using mRNA of 5 day homozygous embryos (the homozygous embryos also show morphological defect),2 whole eyes from 1 year old fish and 2 whole eyes from 3 year old fish which were identified by PCR Screening. Our data consistently show that the mRNA is down regulated in embryos but upregulated in the adult fish. We are not sure how to explain this change.
Any thoughts would be greatly appreciated!
Your question is not completely clear to me and doesn't really seam like an assay problem (as this category of the forum tends to deal with). You might have better success in another cateogyr.
I assume the 1 and 3 year old fish are heterozygotes since the homozygote is lethal by 7 days..so it seems you comparing expression of different genotypes at different ages, and can't really isolate out whether you are looking at an age or genotype effect (or both).
neurobiogal on Fri Nov 19 20:18:37 2010 said:
We have a very puzzling situation in our lab and hopefully someone can help us!
We have identified a point mutation that causes a truncated form of the resulting protein. We know that the mutation is homozygous lethal and zebra fish embryos will die after 7 days and the heterozygous form is viable. I have been performing qPCR using 5 day embryos, 1 year old adults and 3 year old adults. I have run real time using mRNA of 5 day homozygous embryos (the homozygous embryos also show morphological defect),2 whole eyes from 1 year old fish and 2 whole eyes from 3 year old fish which were identified by PCR Screening. Our data consistently show that the mRNA is down regulated in embryos but upregulated in the adult fish. We are not sure how to explain this change.
Any thoughts would be greatly appreciated!