Gene silencing in mammalian cells by preformed small RNA duplexes☆
Section snippets
Materials and methods
Chemically synthetic siRNAs against the protein kinase α (PKCα) and oligonucleotides were synthesized in an automatic synthesizer (EUROGENTEC, Belgium). In vitro transcribed siRNAs were synthesized from DNA template oligonucleotides using the T7 RNA polymerase as described by Milligan and Uhlenbeck [6]. For long double-stranded transcripts, the overlapping strategy was used [7]. After transcription, RNAs were purified by 15% polyacrylamide gels containing 7 M urea and stored at −70 °C until use.
Inhibition of PKCα gene expression by chemically and in vitro transcribed siRNAs
Previously, we have reported that active RNA ribozymes can be generated by an in vitro transcription strategy [8]. In this study, we have investigated whether in vitro made 21-nt siRNAs would function like their chemically synthetic counterparts [5]. Because the first nucleotide incorporated into the RNA by T7 RNA polymerase is a guanosine [6], in vitro transcribed siRNAs must start with at least one guanosine. We designed various gene-specific siRNAs targeting the protein kinase Cα and GFP (
Discussion
The ability of siRNA to interfere with gene expression in a specific manner is the basis for the therapeutic potential of these molecules especially in diseases where the production of aberrant proteins as well as the overexpression of normal proteins is a predominant feature. In the present study, we show that PKCα and GFP gene expression can be silenced by in vitro transcribed and chemically made siRNAs in a sequence-specific manner. Importantly, we show that long double-stranded siRNA with
Acknowledgements
This research was supported by the Norwegian Cancer Society. We thank Drs. Anne Dybwad and Øyvind Melien for their comments on the manuscript.
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Abbreviations: siRNA, small interfering RNA; GFP, green fluorescent protein; PKC, protein kinase C; RNAi, RNA interference.