Hi, has anyone exprience, the luciferase assay resulted in increasing the expression but by the analysis using real-time it is low/no expression at all. Is it because post-transcriptional regulation? For the time being, I was only using one time course (24 hour treatment of my sample on HepG2 cell). I'm planning to do several hours to see the pattern. Does luciferase assay correlate with real-time PCR? Thank you in advance.
The result obtained in RT-PCR will give you not the amount of mRNA that is being created but the amount of mRNA that you are creating minus the mRNA that is being degradated, maybe your mRNA is suffering an early degradation you can look for some proteins which stabilize the mRNA such as HuR. Also the miRNA might also be getting rid of your mRNA
Luc assay measures your POI's promoter activity in an artificial cell system. Your POI even does not expressed in Hep cells. So I do not really understand why you want to measure RT-PCR. Mightbe I went wrong somewhere.
Roberto : Thank you for the idea. I'll try to find about it.
Attila: I did luciferase screening for my sample so that whichever sample express more than control, i proceed with real-time PCR to measure the mRNA levels. But I want to know, is there any correlation between those two (Luc and RT-qPCR)?
Promoter of your POI surely works for more different proteins. This means that pos Luc signal will not essentially represent the presence of your target at mRNA level. So you will not get result by qRT-PCR. Good luck!
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