Protocol

Therapeutic Oligonucleotides

Volume 764 of the series Methods in Molecular Biology pp 183-197

Date:

Quantification of siRNAs In Vitro and In Vivo

  • Angie ChengAffiliated withMolecular and Cell Biology Division, Life Technologies
  • , Alexander V. VlassovAffiliated withMolecular and Cell Biology Division, Life Technologies Email author 
  • , Susan MagdalenoAffiliated withMolecular and Cell Biology Division, Life Technologies

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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.

Key words

siRNA shRNA miRNA RNAi quantification PCR TaqMan® siRNA assays