Too much interference: injection of double-stranded RNA has nonspecific effects in the zebrafish embryo

Dev Biol. 2000 Aug 1;224(1):20-8. doi: 10.1006/dbio.2000.9761.


We have investigated the ability of double-stranded RNA (dsRNA) to inhibit gene expression in a vertebrate, the zebrafish, Danio rerio. Injection of dsRNA corresponding to the T-box gene tbx16/spadetail (spt) into early wild-type embryos caused a rapid and dramatic loss of tbx16/spt mRNA in the blastula. mRNAs from the papc, tbx6, and gata1 genes, which depend on tbx16/spt function for their expression, were reduced, apparently mimicking the spt mutant phenotype. However, mRNAs from a number of genes that are unaffected by the spt mutation, such as beta catenin, stat3, and no tail, were also lost, indicating that the "interference" effect of tbx16/spt dsRNA was not restricted to the endogenous tbx16/spt mRNA. We compared the effects of injecting dsRNA from the zebrafish tbx16/spadetail, nieuwkoid/bozozok, and Brachyury/no tail genes with dsRNA from the bacterial lacZ gene. In each case the embryos displayed a variable syndrome of abnormalities at 12 and 24 h postfertilization. In blind studies, we could not distinguish between the effects of the various dsRNAs. Consistent with a common effect of dsRNA, regardless of sequence, injection of dsRNA from the lacZ gene was likewise effective in strongly reducing tbx16/spt and beta catenin mRNA in the blastula. These findings indicate that, despite published reports, the current methodology of double-stranded RNA interference is not a practical technique for investigating zygotic gene function during early zebrafish development.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Embryo, Nonmammalian
  • Gene Silencing
  • In Situ Hybridization
  • Microinjections
  • Phenotype
  • RNA, Double-Stranded / genetics*
  • RNA, Messenger / analysis
  • T-Box Domain Proteins / genetics
  • T-Box Domain Proteins / metabolism*
  • Zebrafish / embryology
  • Zebrafish / genetics*
  • Zebrafish Proteins*


  • RNA, Double-Stranded
  • RNA, Messenger
  • T-Box Domain Proteins
  • Zebrafish Proteins
  • tbx16 protein, zebrafish