Abstract
RNA interference (RNAi) is a regulatory mechanism of eukaryotic cells that uses small interfering RNAs (siRNA) to direct homology-dependent control of gene activity. Applications of RNAi include functional genomics, in vivo target validation, and gene-specific medicines. A key to in vivo application of siRNA is the advancement of efficient delivery to organs, tissues, or cell types of interest. There is a need to develop reliable and easy-to-use assays to evaluate siRNA delivery efficiency and distribution, study pathways, and stability of siRNAs in cells (post-transfection) and in animals (post- injection). We have adopted the Applied Biosystems TaqMan® based stem–loop RT-PCR technology, originally developed for quantification of endogenous microRNAs in cells, to fulfill these needs. In this chapter, application protocols are described, which enable robust quantification of siRNA, including chemically modified molecules, in vitro and in vivo.
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Acknowledgments
The authors would like to acknowledge the work of C. Chen, Y. Liang, Y. Wang, and L. Wong on the development of TaqMan® siRNA assays and for helpful discussions. Hydrodynamic tail vein injections were conducted under a license grant from Mirus Bio Corporation.
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Cheng, A., Vlassov, A.V., Magdaleno, S. (2011). Quantification of siRNAs In Vitro and In Vivo. In: Goodchild, J. (eds) Therapeutic Oligonucleotides. Methods in Molecular Biology, vol 764. Humana Press. https://doi.org/10.1007/978-1-61779-188-8_12
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DOI: https://doi.org/10.1007/978-1-61779-188-8_12
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