problem in reporducing my result!!! - miRNA expression (Nov/09/2010 )
I have establish a screening strategy to fish out those miRNAs that target my gene. I have fused the 3"UTR (of my gene of interest) to EGFP and expressed under CMV promoter. On the other hand, I cloned the pri-miRNA (flanked with 250bp)into the intron of dsRED (expressed under CMV promoter). So, I am able to use dsRED as the indication of miRNA expression and EGFP as my reporter. This systme work well and I verified my result with luciferase assay and WB. The problem is, since last month, I failed to reproduce the result. Everything I used is the same, same plasmid same 293T cells, same reagent. There is no downregulation anymore for my positive control. Could anyone help me to troubleshoot???
Thanks a lot
Are your lentiviral particles previously or newly packaged? Either could introduce variation in viral activity, resulting in inconsistence in results.
Did you get similar mRNA expression (similar dsRed signal)?