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Assays for Direct and Indirect Effects of C. elegans Endo-siRNAs

  • Philip K. Shiu
  • Jimmy J. Zhuang
  • Craig P. HunterEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1173)

Abstract

Ever since the discovery of the first microRNAs in C. elegans, increasing numbers of endogenous small RNAs have been discovered. Endogenous siRNAs (endo-siRNAs) have emerged in the last few years as a largely independent class of small RNAs that regulate endogenous gene expression, with mechanisms distinct from those of piRNAs and miRNAs. Quantification of these small RNAs and their effect on target RNAs is a powerful tool for the analysis of RNAi; however, detection of small RNAs can be difficult due to their small size and relatively low abundance. Here, we describe the novel FirePlex assay for directly detecting endo-siRNA levels in bulk, as well as an optimized qPCR method for detecting the effect of endo-siRNAs on gene targets. Intriguingly, the loss of endo-siRNAs frequently results in enhanced experimental RNAi. Thus, we also present an optimized method to assess the indirect impact of endo-siRNAs on experimental RNAi efficiency.

Keywords

siRNA quantification Endo-siRNA targets RNAi efficacy FirePlex assay qPCR 

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Philip K. Shiu
    • 1
  • Jimmy J. Zhuang
    • 1
  • Craig P. Hunter
    • 1
    Email author
  1. 1.Department of Molecular and Cellular BiologyHarvard UniversityCambridgeUSA

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