Protocol

RNA-Protein Interaction Protocols

Volume 488 of the series Methods in Molecular Biology pp 367-381

Designing and Utilization of siRNAs Targeting RNA Binding Proteins

  • Dong-Ho KimAffiliated withMolecular Biology Division, Beckman Research Institute of the City of Hope
  • , Mark BehlkeAffiliated withIntegrated DNA Technologies (IDT)
  • , John J. RossiAffiliated withMolecular Biology Division, Beckman Research Institute of the City of Hope

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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