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Protein shows KD but RNA level is higher


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#1 zodiac1505

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Posted 13 August 2010 - 05:54 AM

I am trying to knock down a protein in cell lines. I have tested 9 shRNA sequences so far and all of them showed good KD in luciferase assay but once I did western blot with the infected cells, there was no knockdown. Now I am testing the 9th sequence and saw a reduction in the protein level by western blot. But the RNA level seems to be almost twice compared to the control(qPCR) . I read about translational repression. Anyone could think of some explanation? I have already spent 2 and half years of my life trying to get this project to work <_< Forgot to add one thing. I have the shRNA in a vector with the mir30 motif

Edited by zodiac1505, 13 August 2010 - 06:42 AM.


#2 Jon Moulton

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Posted 13 August 2010 - 06:29 AM

We see a similar effect sometimes with steric-blocking antisense: increased mRNA, decreased protein product.

If a gene is under feedback inhibition and translation if the gene product is inhibited by an oligo, transcription will be upregulated. If (1) your siRNAs are not degrading their target RNA but are inhibiting the translation of that RNA and (2) the gene is regulated by feedback inhibition, then an increase in the RNA level and a decrease in the protein level would be a reasonable outcome.

Another possibility is that the inhibition of translation also protects the mRNA from nucleolytic activity. That way the RNA could build up by persisting longer and the observed increased RNA and decreased protein could occur without feedback regulation of transcription.
Jon D. Moulton
Gene Tools, LLC
www.gene-tools.com

#3 zodiac1505

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Posted 13 August 2010 - 06:48 AM

We see a similar effect sometimes with steric-blocking antisense: increased mRNA, decreased protein product.

If a gene is under feedback inhibition and translation if the gene product is inhibited by an oligo, transcription will be upregulated. If (1) your siRNAs are not degrading their target RNA but are inhibiting the translation of that RNA and (2) the gene is regulated by feedback inhibition, then an increase in the RNA level and a decrease in the protein level would be a reasonable outcome.

Another possibility is that the inhibition of translation also protects the mRNA from nucleolytic activity. That way the RNA could build up by persisting longer and the observed increased RNA and decreased protein could occur without feedback regulation of transcription.



Thanks a lot for the reply. I was trying to read up on this and saw that with microRNAs, translational repression is the case. Does it usually happen with shRNAs or is it more common for shRNAs to degrade the mRNA? My construct has a mir30 motif. Does that matter? Also the protein is a tumour suppressor. Should I be worried that I can't see a reduction at the RNA level? :huh:

Edited by zodiac1505, 13 August 2010 - 06:49 AM.


#4 Jon Moulton

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Posted 16 August 2010 - 07:37 AM

Molecules using Argonaute (e.g. RNAi, siRNA, miRNA, shRHA) can repress translation of mRNA. I don't know how the miR30 motif will affect your system, though I wonder if the target mRNA has an miR30 target. As far as worrying that you cannot see a decrease in mRNA, I suppose that depends on what outcome you are trying to achieve. If you want to knock down the activity of the protein and you have repressed the translation of the protein enough that the band has mostly disappeared from an immunoblot, then perhaps you have reached your goal. Perhaps you have a mispair or two between your shRNA and your targeted mRNA; that is more likely to lead to translation repression rather than mRNA degradation. Did you determine the target sequence in your experimental strain or cell line, or are you working from a species-specific database?

Here's an old but good paper - thank you Dr. Collins.

Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Scacheri PC, Rozenblatt-Rosen O, Caplen NJ, Wolfsberg TG, Umayam L, Lee JC, Hughes CM, Shanmugam KS, Bhattacharjee A, Meyerson M, Collins FS. Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1892-7. Epub 2004 Feb 09.
Jon D. Moulton
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www.gene-tools.com

#5 zodiac1505

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Posted 16 August 2010 - 11:13 PM

Molecules using Argonaute (e.g. RNAi, siRNA, miRNA, shRHA) can repress translation of mRNA. I don't know how the miR30 motif will affect your system, though I wonder if the target mRNA has an miR30 target. As far as worrying that you cannot see a decrease in mRNA, I suppose that depends on what outcome you are trying to achieve. If you want to knock down the activity of the protein and you have repressed the translation of the protein enough that the band has mostly disappeared from an immunoblot, then perhaps you have reached your goal. Perhaps you have a mispair or two between your shRNA and your targeted mRNA; that is more likely to lead to translation repression rather than mRNA degradation. Did you determine the target sequence in your experimental strain or cell line, or are you working from a species-specific database?

Here's an old but good paper - thank you Dr. Collins.

Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Scacheri PC, Rozenblatt-Rosen O, Caplen NJ, Wolfsberg TG, Umayam L, Lee JC, Hughes CM, Shanmugam KS, Bhattacharjee A, Meyerson M, Collins FS. Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):1892-7. Epub 2004 Feb 09.



Thanks a lot for the paper. I read up on my protein and it seems what you said earlier makes sense. My protein is in deed regulated by feedback inhibition. All I want is the protein to be gone and I do see a reduction in the WB. But what I don't understand is why the other 8 shRNAs failed to knock down the protein. The one I used now is a TRC clone that has already been published and this seems to work.




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