Validating siRNA using a reporter made from synthetic DNA oligonucleotides

Quan Du, Håkan Thonberg, Hong Yan Zhang, Claes Wahlestedt, Zicai Liang

Research output: Contribution to journalArticle

34 Scopus citations

Abstract

Only a small fraction of all siRNAs are effective in silencing their target genes, and siRNA efficacy can only be determined experimentally. Previously described reporter-based siRNA validation methods all rely on the availability of physical cDNA clones, and this limits the high throughput applicability of the method. In the current report, we used short synthetic DNA fragment containing a siRNA targeting site, instead of cDNA, to fuse with a reporter gene. When targeting such transcripts with different siRNAs, we found that such constructs can faithfully report the efficacy of the corresponding siRNAs in a sequence specific manner, even when the inserted DNA fragment is essentially only long enough to cover the targeting site. The efficacy of both vector-based siRNA and synthetic siRNA can be evaluated using this system. Since only readily available short synthetic DNA fragments are needed for forming the evaluation vector, this method provides an appealing way of validating siRNAs in high throughput.

Original languageEnglish (US)
Pages (from-to)243-249
Number of pages7
JournalBiochemical and biophysical research communications
Volume325
Issue number1
DOIs
StatePublished - Dec 3 2004
Externally publishedYes

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Keywords

  • Dual-luciferase assay
  • Efficacy validation
  • Oligonucleotides
  • Reporter
  • siRNA
  • Target site

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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