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Thread: shRNA question

  1. #1 shRNA question 
    Forum Masters Degree samcdkey's Avatar
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    What are the conditions under which treatment of cells with shRNA would increase the expression of a target gene instead of silencing it?


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  3. #2  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    When the shRNA inhibits the inhibitor of your fav gene.


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    shRNA activates RNA interference (RNAi), which is involved in gene silencing.


    The RNAi pathway is found in many eukaryotes including animals and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA molecules into short fragments of ~20 nucleotides. One of the two strands of each fragment, known as the guide strand, is then incorporated into the RNA-induced silencing complex (RISC). The most well-studied outcome is post-transcriptional gene silencing, which occurs when the guide strand base pairs with a complementary sequence of a messenger RNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex. This process is known to spread systemically throughout the organism despite initially limited molar concentrations of siRNA.
    So I'd guess that errors in cleavage could halt the silencing process among other things.
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  5. #4  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    She found elevated levels of expression when adding the shRNA (or so I speculate from her post)

    That cannot be explained from faulty inhibition.


    Then it would just go to a normal level.
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    If you treat cells with mutated shRNA couldn't it lead to problems in cleavage? That would be "a condition under which treatment of cells with shRNA would increase expression of a target gene" ..or would a mutation in shRNA more likely cause something else to happen?
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  7. #6  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    I am just assuming samcdkey was using proper scientific language here, and with that I would dismiss your theory.
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    Quote Originally Posted by spuriousmonkey
    I am just assuming samcdkey was using proper scientific language here, and with that I would dismiss your theory.
    Please explain...as I am not as experienced as you are...
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  9. #8  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    What are the conditions under which treatment of cells with shRNA would increase the expression of a target gene instead of silencing it?
    I know she is a scientist. So when she says 'treatment of cells' I know she had at least two samples.

    One control sample: untreated.

    One experiment sample: treated.

    She apparently added the shRNA to a sample of cells. She expected an inhibition of her favourite gene (the one she is looking at).

    She found elevation of the gene expression, as compared to the control sample. Elevated means the expression levels were way above normal (as in the control sample).

    The simplest explanation is that the shRNA cancelled an inhibition. So i suggested that the shRNA actually inhibited an inhibitor of her gene of question.

    Naturally being a good scientist, she actually had three samples.
    1. control: nothing added.
    2. control: scramble shRNA added.
    3. experiment: specific shRNA added.

    And if it is all a new approach she would have a fourth positive control.
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    Thanks! *Pets spurious George* :P
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  11. #10  
    Forum Masters Degree samcdkey's Avatar
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    Its not my study, but the sequence of events as defined by spurious is correct.

    Unfortunately, the master demands that repeated experiments demonstrating the same result reveal a hitherto hidden tendency to extreme stupidity. Compounded by the absence of knowledge regarding a known inhibitor of said gene, the current dogma is to repeat until desired results are obtained, otherwise known as the definition of insanity.

    Meanwhile, two months have passed in futile repetition.

    Drama aside, what is the best way to substantiate this? Note that this is the first time working with shRNA and there was a steep learning curve
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  12. #11  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    I think the solution is very simple.

    You need to design a set of shRNAs for your target and test them all. Not just one.

    One can fail. All cannot.

    If there is still elevation of expression with all independently designed shRNAs you do not have a problem: you have a Nature paper in your hands.

    That is if you can find out why.

    I assume you are using a cell line? (tsk).
    I assume you put a construct in that cell line? (tsk)
    Repeat the experiment with another cell line.

    Try to eliminate all variables.

    It will take you yonks and yonks, and maybe the conclusion is that you shouldn't bother to start with. Because if the experiment isn't vital then it is simply a waste of time.

    The way you described events sounds familiar. I heard recently of a case where a qPCR needed to be repeated until the 'desired' results were obtained, obviously dismissing a whole set of negative results as insignificant.

    This kind of approach to science is often pushed by groupleaders and I don't think it is the right approach. There are reasons why some experiments do not work. One of which is that maybe your conceptions on what is happening is false. But that possibility is mostly overlooked, and instead people blame incompetence of the people doing the experiment or technological difficulties.

    I also noticed that it often takes a long long time to convince the person in charge that there is a possibility that they could be just wrong in their thinking. And often this isn't a possibility at all.

    So if you have not done it already:

    1. Set of specific shRNAs.
    2. Different cell line.
    3. Drop the experiment, and design a better one.
    4. And I hope that you didn't forget to check both the messenger levels and the protein levels.

    That's all I can suggest based on the limited data.

    On a side note: for miRNAs it is extremely common that they target multiple genes, up to hundreds. I recently found a case where one actually targeted multiple genes in a single signaling family.

    I'm actually doing a paper on miRNA myself nowadays.
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  13. #12  
    Forum Masters Degree samcdkey's Avatar
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    Three cell lines have been covered in the futile repetition. Apparently all this proves is "you don't know anything", as the scientist is evidently a coach and rah rah encouragment of cells in the right manner is conducive to generating the desired results.

    Unfortunately, this is but a small part of a larger cooperative effort, involving ego and money, so failure is not an option.

    Don't know if set of shRNAs have been covered. I kinda assumed they must have been.

    Thanks for the help.
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  14. #13  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    Never assume anything.

    I recently assumed they knew what was expressed in the modified gene of a natural mutation, since they had several papers on it, one in PNAS.

    Turns out nobody knew.

    Despite more than a decade of work on it.

    pffft.
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  15. #14  
    Forum Masters Degree samcdkey's Avatar
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    My assumption was based on my experience with the person doing the study, who is the epitome of "leaves no stone unturned"
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  16. #15  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    So did this person check mRNA and protein levels?
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  17. #16  
    Forum Masters Degree samcdkey's Avatar
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    Thats on the list of questions to ask. We did not have a lot of time to chat.
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  18. #17  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    "Kill them all and let God sort them out."

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  19. #18  
    Forum Masters Degree samcdkey's Avatar
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    Quote Originally Posted by spuriousmonkey
    The friend works in a different state in a different lab.

    We occasionally brainstorm with each other.

    Send me a link to your microRNA paper when its done. ")
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  20. #19  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    That could take a while. We (predictably) run into delays once we started to the in vitro culture part of the paper.

    We are semi on track again. Depends on the results of the western.
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  21. #20  
    Forum Masters Degree samcdkey's Avatar
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    Whenever, I don't actually work with miRNA but reading for my friend has made me interested.

    Okay, my friend has tried several shRNAs and done the mRNA and protein expression.

    The collaborators are doing the other cell lines.

    So it looks like we have to go back to the drawing board.

    PS. Western? aarrrggghhh. Pour your own, or use readymade?
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  22. #21  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    I let one of our technicians do westerns.

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  23. #22  
    Forum Masters Degree samcdkey's Avatar
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    Wise choice let him/her be exposed to the acrylamide
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  24. #23  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    it's just that i am incompetent.
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  25. #24  
    Forum Masters Degree samcdkey's Avatar
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    IMO, thinking and doing are two separate actions and should be mutually exclusive.

    Anyway, back to the question, any suggestions?
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  26. #25  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    None without knowing the details.
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  27. #26  
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  28. #27  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    I suggest your friend looks for a new postdoc place.
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  29. #28  
    Forum Masters Degree samcdkey's Avatar
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    My friend is already considering abandoning science and teaching English to ESL or some such.
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  30. #29  
    Forum Cosmic Wizard spuriousmonkey's Avatar
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    There is no honour in science. Some people handle it better than others.

    Unfortunately.
    "Kill them all and let God sort them out."

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