Protein come from nowhere - we cannot find RNA (Oct/10/2008 )
We got some trouble.
For at least 3 genes we test in cell lines MCF-15 and panC-28.
We cannot identify any clear band on RT-PCR gel, 2 of the 3 genes we even try 3 different pair of primers that amplify different locations. None of them give any identifiable band. Each PCR amplification, we run at least 38 cycles.
The interesting part is the cell lysate from these 2 cell lines produce strong band on Western gel.
OK we may argue that Western antibody may not be specific enough; but 1 of the gene that we try multi-primer pairs we block with 4 different antibodies (on separate gels of course) and all give a strong band with correct size.
Yes, different dishes were used for RNA or protein purification, and some cDNA maybe old (4 months old). We did repeat RT-PCR and Western from different culture, and got same results.
So, can someone help us explain we have fair amount of protein, but we cannot identify RNA through RT-PCR.
What do we miss here?
Where are those proteins coming from?
Any suggestion is welcome
Thanks
For at least 3 genes we test in cell lines MCF-15 and panC-28.
We cannot identify any clear band on RT-PCR gel, 2 of the 3 genes we even try 3 different pair of primers that amplify different locations. None of them give any identifiable band. Each PCR amplification, we run at least 38 cycles.
The interesting part is the cell lysate from these 2 cell lines produce strong band on Western gel.
OK we may argue that Western antibody may not be specific enough; but 1 of the gene that we try multi-primer pairs we block with 4 different antibodies (on separate gels of course) and all give a strong band with correct size.
Yes, different dishes were used for RNA or protein purification, and some cDNA maybe old (4 months old). We did repeat RT-PCR and Western from different culture, and got same results.
So, can someone help us explain we have fair amount of protein, but we cannot identify RNA through RT-PCR.
What do we miss here?
Where are those proteins coming from?
Any suggestion is welcome
Thanks
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Hello,
I'm not 100% sure about this but since protein is being made and to be specific the correct protein is being made I'm thinking that RNA is getting degraded during the process of RUNNING the RT-PCR OR when running a gel.
Try to stop the gel at different times and see if you can track a band at different times. Maybe RNA is degraded at during the process and runs off the gels. So, you are purifying protein seperately so then somewhere in the process of dealing with RNA alone something is going wrong. Maybe try the gel thing.
Please let me knwo what you think?
Thanks!
Good luck!
Could the gene sequence against which you're designing your primers be wrong?
Thank you for reply
OK I forget this. 
Beside these 2 cell lines, there are few more cell lines that we also process.
The other cell lines, it is clear, no RNA no protein, or got protein there is RNA.
We process all cell lines at same time.
The RT-PCR for gene in question works for other cell lines, thus I think we can say our primers work.
Unless, MCF-15 and PanC-28 have multi-mutation at 3 different location (we have 3 pair of primers cover different locations) within a particular gene. If that is true, this could be Nature or Science level finding and publication, and I don't think I am that lucky (keep hoping though 
).
For same cDNA we also run RT-PCR for different genes and run at same gel. The other genes turn out to be fine for these 2 cell lines so I am not so sure about degradation. Beside we repeated culture cells and extracted RNA and protein, and we got similar result every time (I think it has been repeated at least 3 times).
Any suggestion is welcome
Thank you
Could this be a possibility - your RT-PCR is correct, but your gene is redundant? Meaning that your antibody is recognizing protein because it is being transcribed and translated from a different but similar gene? If the proteins made from these two similar genes are approximately the same size then you may not have realized that there is a redundant protein being made. Searching through the databases should help you to decide if this is a possibility.
That is such a good point!