A novel approach for evaluating the efficiency of siRNAs on protein levels in cultured cells
AUTOR(ES)
Wu, Weilin
FONTE
Oxford University Press
RESUMO
An important aim in the post-sequencing age of functional genomics is to translate gene sequences into protein functions. This shift of focus is particularly necessary for a very large number of human genes, referred to as novel genes, where we have no or very rudimentary information about their biochemical functions. Recently, a new method for investigating human gene functions using small interfering RNA molecules (siRNAs) has become available. siRNAs are powerful reagents for post-transcriptional silencing, where mRNA targeted by the siRNAs is degraded in vivo and the level of the encoded protein is reduced. However, the lack of antibodies against proteins encoded by novel genes restricts the general value of siRNAs as functional tools, as only the mRNA levels can be measured for these genes. We report a method that combines measurements of protein levels in cell culture for novel, exogenously expressed genes, with parallel measurements of the endogenous mRNA levels of the same genes. We find that this combinatorial approach correctly predicts siRNAs that efficiently reduce mRNA and protein levels in cultured cells. Furthermore, this method identifies proteins that have a slow turnover, which weakens the value of the RNA interference method as a tool for functional studies of such genes. The described method should prove to be valuable for large-scale functional studies of novel human genes.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=373369Documentos Relacionados
- A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis
- Expressing functional siRNAs in mammalian cells using convergent transcription
- Structural variations and stabilising modifications of synthetic siRNAs in mammalian cells
- siRNAs can function as miRNAs
- DEQOR: a web-based tool for the design and quality control of siRNAs