validation of microRNA target gene
Posted 12 August 2012 - 09:42 AM
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
Posted 12 August 2012 - 01:09 PM
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.
Posted 12 August 2012 - 01:41 PM
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 !
Posted 12 August 2012 - 01:49 PM
Posted 12 August 2012 - 01:55 PM
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
Posted 20 August 2012 - 10:46 PM