Hi everyone,
I really get a weired problem, I used a plasmid transfection cells, the plasmid has a GFP reporter linked with a protein (lets call it A protein) which I was interested, after transfection 48h, I see many many GFP+ cells (almost >70%) under the microscope, and GFP is very bright, so I assume my protein A is also expressed, however, when I mRNA extract the cells just after I checked the GFP, convert mRNA to cDNA, then run pcr, I got the band for A, but compair with none transfection cells' cDNA, A's mRNA was not overexpress, this really feel weird. If I get mRNA overexpress but no protein, there could be many reasons, but now I get A(as it linked with GFP) protein, but no mRNA overexpress.
Yes, please describe in detail how the 2 proteins are linked. Do you mean fused i.e. A and GFP are one single polypeptide? Which is N-terminal and which is C-terminal? And are you absolutely sure that your PCR recognizes both the native protein A mRNA and the one in the construct? Is your conversion of mRNA to cDNA using oligodT primer?
You already have 3 very helpful comments. If you are generating a fusion protein, don't waste your time with RT-PCR and check your expression by WB with a GFP antibody.
"A's mRNA was not overexpressed". If you just did semi-quantitative RT-PCR, sometimes it is hard to tell the mRNA difference. Better to use quantitative. Besides, A's mRNA may be not stable either.
Hi All, Thanks to the answers. For my 2 protein, they were linked by a T2A sequence, looks like: CAG promoter-protein A-T2A-GFP-polyA , T2A function a little bit like IRES, if one express, the other should express. And the primers were used to bind the exon, which is in mRNA part. also my plasmid PCR shows good bands so I think its not plasmids problem. Maybe the mRNA is not stable, Im not sure about this.
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