Full Text (PDF)
New England Biolabs is devoted to basic research in the areas of RNA biology, gene expression, enzymology, parasitology, and bioorganic chemistry. The research goals of the RNA biology division include the discovery of important regulatory RNAs and proteins that act on RNA from different organisms, as well as the development of methods and tools for RNA research.
Our lab, in the RNA Biology Division, focuses on small RNAs involved in transcriptional or posttranscriptional regulation. Our current projects are: (i) to study the mechanism of RNA silencing in mammalian cells conferred by RNAse III-derived complex small interfering RNA (siRNA) mixtures; (ii) to study transcriptional regulation by short RNAs by targeting promoter and other noncoding RNA sequences; and (iii) to develop regulated microRNA/siRNA expression methods using small molecule regulated pol II-based transcription.
www.neb.com
The Technique
Finding potent siRNAs against a specific target is a major task of many laboratories. We have developed enzymatic methods for producing highly complex siRNA mixtures that can be used for target-specific RNA interference (RNAi) in mammalian cells. In our article, we tested the potency of different siRNA mixtures, targeting the same messenger RNA (mRNA) (p53) and compared them to several commercially available single synthetic siRNAs. For this comparison, we developed a reporter method based on the secreted luciferase from Gaussia princeps by incorporating the target sequence in its 3′ untranslated region. We validated the method by comparing the secreted reporter activity in the cell culture medium to real-time PCR measurements of the target mRNA. The results show that all the enzymatically derived siRNA mixtures have higher silencing potency than most of the synthetic single 21-mers. Our study presents a convenient method for testing siRNAs and suggests that targeting multiple sites of the mRNA by siRNA mixtures ensures robust target silencing.
Determination of silencing potency of synthetic and RNase III-generated siRNA using a secreted luciferase assay, p. 599.
