EJBiotechnology logo
Electronic Journal of Biotechnology ISSN: 0717-3458
  © 2011 by Universidad Católica de Valparaíso -- Chile
Vol. 14 No. 2, Issue of April 15, 2011
 


Fig. 2 Expressivity and application of pFila.
(a) pFila consistently produces Fluc and Rluc activities in human-, mouse- and monkey-sourced mammalian cell lines. pFila was gradientlytransfected into the cells stated above and Fluc and Rluc luciferase levels were determined by a DLRTM Assay (Promega). As shown in this graph, Fluc and Rluc activities were in a linear range for all the selected points.
(b) pFila is capable of recapitulating the interaction of miR16 and its known target CCNE1. Wild-type and mutant CCNE1 3’UTRs were sub-cloned into pFila and co-transfected with miR16-1 mimics. Conventional dual luciferase reporter was conducted in parallel to compare their reproducibility. siRNA against Rluc serves as positive control. Rluc with mutant CCNE1 3’UTR was rescued in comparison with pFila-CCNE1-3’UTR-wildtype, indicating that CCNE1 is a direct target of miR16 as previously reported (Wang et al. 2009).
(c) miR16-1 inhibitors were co-transfected with pFila-CCNE1-3’UTR-wildtype or mutant plasmids into Hela cells by different transfection reagents to block endogenous miR16-1. Inhibition efficacy varies with the selected agents, where RNAiMAX achieved the most potent inhibition. It implies that pFila is a sensitive biosensor for functional miRNA profiling.