2 replies to this topic

[Samantha]

Hi,

I have performed dual luciferase reporter asay to check if my microRNA binds to the potential target gene.  I co-transfected the  luciferase reporter wit the 3UTR (psicheck) and my microRNA, but i didnt observe any changes in the renilla/firefly ratio, compared to cotransfection with the vector control of the microRNA. However, when i co-transfected microRNA with the mutated 3'UTR luciferase vector, the ratio increased, compared to the empty vector of miRNA as well as the wild-type form. How to explain this weird result?

a) wild-type luciferase reporter + empty vector : 1
wild-type luciferase reporter +microRNA: 1

c) mutant luciferase reporter +empty vector: 1
d) mutant luciferase reporter + microRNA: 1.3

Samantha

[pcrman]

Luciferase assay especially dual assay is subject to many errors and biases. Combinatory transfection of other nucleic acids such as plasmids or small RNAs makes the interpretation of results more complicated and error prone. The 1.3 fold increase probably is just artifact and doesn't tell anything.

[whitedolphin]

pcrman, on 05 June 2011 - 08:57 AM, said:

Luciferase assay especially dual assay is subject to many errors and biases. Combinatory transfection of other nucleic acids such as plasmids or small RNAs makes the interpretation of results more complicated and error prone. The 1.3 fold increase probably is just artifact and doesn't tell anything.

I also met a problem to explain the Dual-Luciferase:
there are two miRNA binding sites predicted to target at the gene, and mutation seed region of each sites can relieve the repression of miRNA to 3-5 fold of that in wildtype. However, the unexpected result is that double mutation strengthen the repression instead of removing the repression by this miRNA. What's wrong with it?

________|_luc-UTR__|__luc-UTRm1__|__luc-UTRm2__|_luc-UTRm1&2_|
Empty___|_1.000____|____1.018____|____1.166____|____0.985____|
miRNA___|_0.037____|____0.216____|____0.127____|____0.003____|

Any suggestion are appreciated!

Edited by whitedolphin, 23 July 2011 - 08:59 PM.