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stabile transfection, all have high mRNA level, only ~ 50 % have protein


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#1 talbar

talbar

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Posted 02 March 2011 - 06:28 AM

Hi,
I am hoping somebody might have some ideas?

I have performed stabile transfection of a cell line, and picked 23 monoclonal clones. All were tested for mRNA level using TaqMan gene expression assay, and all of them have expression (this cell line has a homozygous deletion of the gene of interest, and the untransfected and the controls (empty vector) shows no expression using real-time pcr). I test the expression using two probes, one in the first exon-exon boundary, and the other in the last exon-exon boundary. From these results it seems like the entire gene is transcribed. The ORF has been sequenced and should be normal.

But, when I perform western blot, only 11 of the 23 actually have the protein. I thought there should be a pretty good correlation between mRNA and protein level?

The RNA has been DNase-treated prior to reverse transcription, so it should not be DNA-contamination which gives the Ct-values.

It does not seem to be a (good) correlation between the Ct-values and the amount of protein seen on western blot. Some clones show really high mRNA expression, but still have no protein expression. The clones are maintained in 50 % of the selective antibiotic. Is it possible that the cells want to silence the introduced gene, but cannot do it pre-transcriptionally because of the selective agent, and therefore does it post-trascriptionally?

Does anyone has any suggestions? I am soon done with my masters, and are getting pretty desperate.. Thank you so much for your time!

#2 talbar

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Posted 02 May 2011 - 11:20 AM

Maybe somebody has a suggestion if I add that I have over-expressed my gene of interest in a cancer cell line, and the hypothesis is that it might be a tumor suppressor gene?

mRNA overexpressed from open reading frames do not have UTR, do they? I try to search the litterature for translational control mechanims which might explain my results, but I find nothing..

Any thought/suggestion for what could be the problem of further work will be appreciated. :rolleyes:

Thank you!




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