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Article
Quantitative Detection of Double-Stranded RNA-Mediated Gene Silencing of Parasitism Genes in Heterodera glycines
Journal of Nematology
  • Serenella A. Sukno, Texas A & M University - College Station
  • Jamie McCuiston, North Carolina State University
  • Mui-Yun Wong, Universiti Putra Malaysia
  • Xiaohong Wang, United States Department of Agriculture
  • Michael R. Thon, Texas A & M University - College Station
  • Richard Hussey, University of Georgia
  • Thomas Baum, Iowa State University
  • Eric Davis, North Carolina State University
Document Type
Article
Publication Version
Published Version
Publication Date
6-1-2007
Abstract
The introduction of a double-stranded RNA (dsRNA) into an organism to induce sequence-specific RNA interference (RNAi) of a target transcript has become a powerful technique to investigate gene function in nematodes and many organisms. Data provided here indicate that the inclusion of 1–2 mM spermidine and 50 mM octopamine and a 24 hr incubation period of nematodes in double-stranded RNA (dsRNA) soaking solutions resulted in a considerable increase in the percentage of nematodes that ingested dsRNA as compared to previous reports. This modified dsRNA soaking method was coupled with quantitative real-time RT-PCR (qRT-PCR) analyses to assess the potential silencing of the Heterodera glycines parasitism gene transcripts Hg-pel-1 and Hg-4E02 that are expressed within the esophageal gland cells of preparasitic H. glycines J2. The Hg-pel-1 transcript was most efficiently silenced with one dsRNA construct (ds267) at the highest dsRNA soaking concentration of 5.0 mg/ml, while the Hg-4E02 transcript was more efficiently silenced at the 2.5 mg/ml dsRNA concentration as compared to 5.0 mg/ml. A dsRNA construct (ds285) complementary to a different sequence within the Hg-pel-1 transcript than construct ds267 induced only minimal silencing of the Hg-pel-1 transcript at 2.5 mg/ml. The results suggest that both dsRNA concentration and sequence relative to the transcript targeted are critical for maximizing potential RNAi effects in parasitic nematodes.
Comments

This article is published as Sukno, Serenella A., Jamie McCuiston, Mui-Yun Wong, Xiaohong Wang, Michael R. Thon, Richard Hussey, Thomas Baum, and Eric Davis. "Quantitative detection of double-stranded RNA-mediated gene silencing of parasitism genes in Heterodera glycines." Journal of nematology 39, no. 2 (2007): 145.

Rights
Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted.
Language
en
File Format
application/pdf
Citation Information
Serenella A. Sukno, Jamie McCuiston, Mui-Yun Wong, Xiaohong Wang, et al.. "Quantitative Detection of Double-Stranded RNA-Mediated Gene Silencing of Parasitism Genes in Heterodera glycines" Journal of Nematology Vol. 39 Iss. 2 (2007) p. 145 - 152
Available at: http://works.bepress.com/thomas-baum/29/