i am doing qRT-PCR and the results show me that i have a very low amounts mRNA of my gene of interest!!!it is almost the same level as my untreated cells (reference control)!any ideas what that might be?my primers do not detect genomic DNA.thank you
i have read that in some cases a fast degraded protein has higher levels of mRNA cause the cell is trying to compensate the difference!The GFP ic at the C-terminus fused so as to the control receptors!
I have GFP Ab.i am doing all my western blots with that,the receptor and the control receptors(normal and depleted) tagged with GFP are detectable but not the depleted one,GFP band is also there(26 kDa) but no protein.I have tagged also the receptor with flag and did the IF and i had no signals for the depleted! i am investigating the possibility that this depleted part plays any role with the expression of the protein.this is why i did the RT PCR to see the mRNA levels!it is a bit complicated.Actually my question is:mRNA is high but the protein is no expressed or better fast degraded,what can i say actually about the depleted part?is it implicated in these 2 things? total lost here!thanks for all your help
i have two probes:one whole receptor and the receptor with a depleted sequence!in western blots i can detect the whole receptor but not the depletion!in LSM microscopy i can see the receptor(tagged with GFP) on the membrane but the depleted Receptor is all over the cytosol the GFP stain!depletion seems so to be fast degraded but RT PCR gives me high amounts of this mRNA even higher from the normal receptor.any ideas?
i have performed RT PCR and i have found for my gene that the mRNA levels are very high.the problem is that the protein is not expressed in the cells!!!any ideas why?the sequencing is ok,the western blots do not detect protein but the RT PCr gave a high amount of mRNA.thank you
Hi, i have tried RT PCR using as an endogenous control gene GAPDH.i did triplicates of an untreated control and of my samples.Now I want to do the relative calculation of this RT PCR.can anyone help me with this?Thank you in advance.