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validation of microRNA target gene


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6 replies to this topic

#1 alia

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Posted 12 August 2012 - 09:42 AM

I am working in project to find a direct terget gene for one of the microRNAs in melanoma.
Firstly. I did in silico prediction for this target gene then I found the target site in 3'UTR for microRNA X .
I did luciferase assay to see if this miR-X is correlated negatively or positevly with mRNA expression for my target gene. then I found this gene is downregulated by microRNA X in melanoma cell line, then I did site mutation to confirm if this a direct target. the first luciferase data was very promising expect the empty vector which is my control it shows a downregulation. which I can't judge if this gene is a target for this microRNA !! I did this experiment 3 times and the empty vector show me a different result but still my target gene downregulated by this miR-X.
my questions !
can I skipped this control and normalized my data to the negative miR-X mimics data !
is there other method can support my luciferase experiment for mRNA expression !
Thanks for help

#2 pcrman

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Posted 12 August 2012 - 01:09 PM

The downregulation of eppty vector may be resulted from off-target effect of the miRNA or a response to the transfection. Strictly speaking, without showing that your miRNA has no effect on a gene lacking its target cannot support your conclusion that the target you found is a bona fide target.

A few questions:
What is your empty vector? luciferase only without the target?
Did your control miRNA mimics also downregulate empty vector?

I believe the standard control for such assay is to mutate the target (especially the seed region) which has been cloned into the 3UTR of luciferase gene to see if the miRNA of interest can still downregulate the luciferase.

#3 alia

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Posted 12 August 2012 - 01:41 PM

thank you for your answer.

Actually I did site directed mutagensis for my target genes by mutate the seed region, Luciferase assay shows miR-X upregualted the mutated target gene.
I used plightswitch_3UTR from SwitchGear Genomics. I used negative mimics as negative control !
my quiestion is if I can skip the data of empty vector and normalized my data according to the negative mimics !

#4 pcrman

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Posted 12 August 2012 - 01:49 PM

I am sorry for the misunderstanding. If you already have the mutated target vector control, then you don't need to have a empty vector control. The mutation of seed sequence abolishes the inhibitory effect by the miRNA supports your conclusion that the target is real.

#5 alia

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Posted 12 August 2012 - 01:55 PM

ah good ! then I can skipped the data of this empty target gene then normalized my data to the negative mimics.
I have now five experiments what is the best calculation way to sum up my data ! is by taking the Median or the average !

Thank you very much

#6 klenow

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Posted 20 August 2012 - 03:26 AM

Another test (but requires some work and maybe is not necessary) is to build a miRNA spongue to your candidate miRNA and check what happens with your candidate gene when the spongue is transfected into cells.

#7 alia

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Posted 20 August 2012 - 10:46 PM

Thanks Kienow. Your method it looks very interesting, but unfortunately, I don't have enough time to do it. Anyway after some statistic's calculation I skipped this empty plasmid (positive control) and just normalized my data to the negative mimics.




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