problem in reporducing my result!!! - miRNA expression (Nov/09/2010 )

HI all,

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

Wen Hao.

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)?