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Unable to get proper PCR amplification


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#1 madrius1

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Posted 05 January 2011 - 02:32 PM

Hi,

We've been having a lot of trouble amplifying our protein of interest with PCRs. We've try designing primers on exon-exon junctions to avoid genomic contamination, we've try a lot of regions on the transcript, and we never got any good results. The main problem is not to get a "clean" amplification, but to get one that correlates with protein quantity. Of course, we are aware that RNA and protein level do not always correlate. But in this case, the RNA gets down, whereas the protein increases. And the 2 antibodies we use to detect our protein both respond to knockdown. The funny thing is that none of the paper talking about this protein have qPCR data..

Do you guys know examples of other proteins whose expression could not be monitored by qPCR?

Do you have any tips/suggestions?

#2 ElHo

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Posted 06 January 2011 - 01:46 AM

Maybe the knockdown acts at the translational level. Then you would not see any effect on the transcriptional level.

#3 HomeBrew

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Posted 06 January 2011 - 03:51 AM

And the 2 antibodies we use to detect our protein both respond to knockdown.


Gene duplication in the genome? I assume the band you're getting with the knockdown samples is indistinguishable from the known protein band... Are your antibodies polyclonal of monoclonal? There could be a gene in the genome, other than the one that's presumably knocked out, that produces the "same" protein. The DNA sequence of this putative second gene need not match the sequence of the one that's knocked out (code degeneracy), and so would not be detected by your PCR, but might compensate for the knockout if they're coordinately regulated.

Have you checked the genome of the knockout to see that it's actually knocked out?

#4 madrius1

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Posted 06 January 2011 - 07:03 AM

Thanks for your inputs.

The translational knockdown make sense, i'll keep that in mind.

And as for the second respond, we do not have a knockout, but a knockdown. :)




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