how to make 3'UTR reporter - (Mar/19/2010 )
Hi I am new. I want to make 3'UTR of target gene reporter to test miRNA of interest. I plan to insert 100bp olig containg miRNA binding sites in to Pmir- reporter. Should I design the oligo same as the sequence of 3'UTR sequence I get from NCBI database, or the complementary sequence of 3'UTR? I am confused , need help and thanks in advance.
Your insert should be the same sequence as the cDNA sequence you got from NCBI database, no need to reverse-complement it.
If you want to examine a (potential) miRNA and its expression, you can clone the predicted mature miRNA antisense sequence into a reporter vector.
I have found this site used the reporter assay to validate miRNA expression, it's more like miRNA serves as siRNA assay.
If you want to examine a miRNA target messenger RNA, then you might want to clone the 3'UTR of the target gene into a reporter vector.