RNA-Protein Interaction Protocols pp 367-381

Part of the Methods in Molecular Biology book series (MIMB, volume 488)

Designing and Utilization of siRNAs Targeting RNA Binding Proteins

  • Dong-Ho Kim
  • Mark Behlke
  • John J. Rossi

Summary

Small interfering RNA (siRNA)-mediated RNA interference (RNAi) is a very powerful tool for triggering posttranscriptional gene silencing in several organisms. We discuss the improvement of two different sources of siRNAs synthesized either chemically or by an enzymatic method. When the siRNAs are synthesized by in vitro transcription using a phage polymerase, the initiating triphosphates trigger a potent interferon induction that can lead to misinterpretation of the data. A novel method is presented to minimize the nonspecific effect of enzymatic siRNAs while maintaining the advantages of lower cost and less turnaround time. When chemical siRNAs are used, the expense and long turnaround time can be a problem, especially if the selected siR-NAs are not highly functional in triggering RNAi. The new format for making double-stranded RNAs (dsRNAs) is described to achieve more efficient suppression. The format has been tested by creating siRNAs targeting two RNA binding proteins, La and hnRNP (heterogeneous nuclear ribonucleoprotein) H, and has shown better potency at lower concentrations than the conventional 21-mer siRNA.

Key Words

Dicer Dicer substrates in vitro transcription RNA RNA interference siRNA triphosphate 

Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Dong-Ho Kim
    • 1
  • Mark Behlke
    • 2
  • John J. Rossi
    • 3
  1. 1.Molecular Biology DivisionBeckman Research Institute of the City of HopeDuarteUSA
  2. 2.Integrated DNA Technologies (IDT)CoralvilleUSA
  3. 3.Molecular Biology DivisionBeckman Research Institute of the City of HopeDuarteUSA

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