Testing the putative effect of Dicer-substrate siRNAs in regulating gene expression at transcriptional level

Authors : Müge KOVANCILAR, Mehmet Nejat DALAY, Uğur DELİGEZER

Pages : 399-404

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Publication Date : 2011-12-01

Article Type : Research Paper

Abstract :RNA interference insert ignore into journalissuearticles values(RNAi); pathway is a gene silencing process during which small double-stranded RNA insert ignore into journalissuearticles values(dsRNA); molecules trigger the degradation of homologous RNA targets. Small interfering RNAs insert ignore into journalissuearticles values(siRNAs); are the mediators of the RNAi that can be induced in vitro and in vivo by direct application of chemically synthesized siRNAs. Recently, promoter-targeted small non-coding RNAs have been described to be capable of regulating gene expression at the transcriptional level. In the present study we tested the hypothesis that Dicer-substrate siRNAs insert ignore into journalissuearticles values(D-siRNAs);, which trigger gene silencing through intrinsic RNAi pathway and are therefore more potent in gene knockdown than traditionally used 21-23mer siRNAs, may also display regulatory effects at the transcriptional level. Synthetic 27mer D-siRNAs targeting the promoter/early coding region of the CDKN2A gene were designed and transfected into HeLa cells, and 48 h post-transfection the CDKN2A expression was analyzed in quantitative real-time PCR using the house-keeping G6PDH gene as reference. In comparison to the mutant version, the CDKN2A gene was effectively knocked-down by the D-siRNA, but no evidence of transcriptional regulation was found. We conclude that the D-siRNA-induced suppression is likely to occur after transcription.
Keywords : Key words RNA interference, Dicer substrate siRNAs, transcriptional silencing, CDKN2A