RNAi

"RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene." Tuschl et al. Nature Vol. 41, 2001

Small-interfering RNAs (siRNA) have been generated for purposes of specific knock-down studies of endogenous genes in mammalian cells. Based on studies of RNAse III activities in response to large double stranded RNA during viral replication, it has been found that 21-nucleotide RNAs mediate RNAi in cultured mammalian cells through a process of targeted mRNA degredation that is highly effective at nM concentrations.

We have utilized these technologies for specific knock-down studies to evaluate the necessity of enzymes of sphingolipid metabolism. For more on designing dsRNA oligos, see: http://www.mpibpc.gwdg.de/abteilungen/100/105/sirna_u.html.

Cells were transfected with Oligofectamine according to manufactures instructions, and evaluated at a time point dependent on the stability of the protein of interest. We found 48 hours post oligofection (50% confluency) at a dsRNA concentration of 200nM to be optimal for most enzymes of interest.

Benjamin J. Pettus

References

Elbashir, S. M., et al. (2001a). Duplexes of 21-nucleotide RNAs mediate RNA interference in mammalian cell culture. Nature 411: 494-498.

Elbashir, S. M., W. Lendeckel and T. Tuschl (2001b). RNA interference is mediated by 21 and 22 nt RNAs. Genes & Dev. 15: 188-200.

Elbashir, S. M., J. Martinez, A. Patkaniowska, W. Lendeckel and T. Tuschl (2001c). Functional anatomy of siRNAs for mediating efficient RNAi in

Drosophila melanogaster embryo lysate. submitted.

Harborth, J. et al. (2001). Identification of essential genes in cultured mammalian cells using small interfering RNAs. J. Cell Science 114: 4557-4565.

Masters, J. R., et al. (2001). Short tandem repeat profiling provides an international reference standard for human cell lines. Proc. Natl. Acad. Sci.

USA 98: 8012-8017.

Tuschl, T., P. D. Zamore, R. Lehmann, D. P. Bartel and P. A. Sharp (1999). Targeted mRNA degradation by double-stranded RNA in vitro. Genes &

Dev. 13: 3191-3197.