3 replies to this topic

[NDG]

Hi,

I performed an absolute quantification of ULBP2 mRNA for my cancer cell lines.I also did a flow analysis on them to know the protein expression on the surface.Flow cytometry shows some of them to have MFI values between 800 and 900.However, my qPCR shows extremely low mRNA levels.In fact the maximum I get is 2 copies/cell.Most of the cells that are positive for my protein by flow, show almost 0 copies of mRNA.Is that possible?

Can a cell have no mRNA but express the protein on the surface.These are steady state readings.

Any help will be greatly appreciated.Thanks

NDG

Edited by NDG, 08 July 2009 - 04:34 PM.

[gfischer]

NDG, on Jul 8 2009, 07:33 PM, said:

Hi,

I performed an absolute quantification of ULBP2 mRNA for my cancer cell lines.I also did a flow analysis on them to know the protein expression on the surface.Flow cytometry shows some of them to have MFI values between 800 and 900.However, my qPCR shows extremely low mRNA levels.In fact the maximum I get is 2 copies/cell.Most of the cells that are positive for my protein by flow, show almost 0 copies of mRNA.Is that possible?

Can a cell have no mRNA but express the protein on the surface.These are steady state readings.

Any help will be greatly appreciated.Thanks

NDG

Either your primers are no good, or you're getting false positive staining with the antibodies.  Go back and validate them both to make sure they work properly.

[Rsm]

Hi,
That is indeed possible. It has been shown for mouse hematopoietic stem cells that they do not express CD34 on the surface, but have a lot of CD34 mRNA in them... Well it's the other way round, but it sure does not correlate. Anyway, I would expect surface proteins to have a rather long half-life, so it might be sufficient to have mRNA synthesis low while maintaining a good expression on the surface.
Just my 2 cents...

Cheers,
Minna

[ivanbio]

Hi NDG,

Your situation sounds entirely possible. I do not know what is the lifespan of ULBP2 mRNA or protein, but here is a simple explanation for your results: ULBP2 protein could be quite stable so it could be synthesized from its mRNA, which could be quite unstable, early on. By the time you analyze for the presence of ULBP2 mRNA, the mRNA could be completely gone and there is plenty of protein present that would last for a long time after the mRNA is gone.

My $0.02