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one target gene - RT-PCR: upregulation, Western Blot: no overexpression

one target gene - RT-PCR: upregulation, Western Blot: no overexpression - Real-Time PCR and Quantitative PCR Forum

one target gene - RT-PCR: upregulation, Western Blot: no overexpression - Real-Time PCR and Quantitative PCR Forum


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Old 12-16-2011, 11:16 AM
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Default one target gene - RT-PCR: upregulation, Western Blot: no overexpression



Hi!

According to central dogma of molecular biology protein synthesis requires 2 steps: transcription and translation.

What if there is transcription of the target gene after stress impulse but none of translation of the protein coded by the target gene?

Quantitative RT-PCR showed upregulation of the target gene but Western Blot did not show any upregulation of the protein coded by the target gene.

How can it be interpreted?

Thank you.
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Old 12-16-2011, 01:55 PM
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Default Re: one target gene - RT-PCR: upregulation, Western Blot: no overexpression

Quote:
Originally Posted by Kobrus View Post
Hi!

According to central dogma of molecular biology protein synthesis requires 2 steps: transcription and translation.

What if there is transcription of the target gene after stress impulse but none of translation of the protein coded by the target gene?

Quantitative RT-PCR showed upregulation of the target gene but Western Blot did not show any upregulation of the protein coded by the target gene.

How can it be interpreted?

Thank you.
The gene could be regulated post-transcriptionally. Translation of RNA is dependent upon the presence of a polyA tail and there are known instances of regulation via cytoplasmic addition of polyA tracts.

Also micro-RNAs and RNA binding proteins can repress translation of RNA.

Another possibility is that the RNA is translated, but the protein is rapidly degraded so that when you look at steady state protein by western blot, you see no change.

To test if the RNA is being translated you could determine if it is associated with polysomes.
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Old 12-16-2011, 03:36 PM
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Default Re: one target gene - RT-PCR: upregulation, Western Blot: no overexpression

Thanks for reply!

How to test if the mRNA is associated with polysomes?!
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blot , gene , overexpression , rtpcr , target , upregulation , western


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