• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of rnaThe RNA SocietyeTOC AlertsSubscriptionsJournal HomeCSHL PressRNA
RNA. Nov 2002; 8(11): 1454–1460.
PMCID: PMC1370351

Functional siRNA expression from transfected PCR products.

Abstract

RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) induces the postranscriptional degradation of homologous transcripts. RNAi can be initiated by exposing cells to dsRNA either via transfection or endogenous expression. In mammalian systems, the sequence-specific RNAi effect has been observed by expression of 21-23 base transcripts capable of forming duplexes, or via expression of short hairpin RNAs. We describe here a facile PCR based strategy for rapid synthesis of siRNA expression units and their testing in mammalian cells. The siRNA expression constructs are constructed by PCR, and the PCR products are directly transfected into mammalian cells resulting in functional expression of siRNAs. This approach should prove useful for identification of optimal siRNA-target combinations and for multiplexing siRNA expression in mammalian cells.

Full Text

The Full Text of this article is available as a PDF (1.5M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature. 2001 Jan 18;409(6818):363–366. [PubMed]
  • Bernstein E, Denli AM, Hannon GJ. The rest is silence. RNA. 2001 Nov;7(11):1509–1521. [PMC free article] [PubMed]
  • Brummelkamp Thijn R, Bernards René, Agami Reuven. A system for stable expression of short interfering RNAs in mammalian cells. Science. 2002 Apr 19;296(5567):550–553. [PubMed]
  • Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001 May 24;411(6836):494–498. [PubMed]
  • Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001 Jan 15;15(2):188–200. [PMC free article] [PubMed]
  • Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science. 2001 Aug 10;293(5532):1146–1150. [PubMed]
  • Holen Torgeir, Amarzguioui Mohammed, Wiiger Merete T, Babaie Eshrat, Prydz Hans. Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. Nucleic Acids Res. 2002 Apr 15;30(8):1757–1766. [PMC free article] [PubMed]
  • Ketting RF, Fischer SE, Bernstein E, Sijen T, Hannon GJ, Plasterk RH. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 2001 Oct 15;15(20):2654–2659. [PMC free article] [PubMed]
  • Lee Nan Sook, Dohjima Taikoh, Bauer Gerhard, Li Haitang, Li Ming-Jie, Ehsani Ali, Salvaterra Paul, Rossi John. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol. 2002 May;20(5):500–505. [PubMed]
  • McManus Michael T, Petersen Christian P, Haines Brian B, Chen Jianzhu, Sharp Phillip A. Gene silencing using micro-RNA designed hairpins. RNA. 2002 Jun;8(6):842–850. [PMC free article] [PubMed]
  • Miyagishi Makoto, Taira Kazunari. U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol. 2002 May;20(5):497–500. [PubMed]
  • Moss EG. RNA interference: it's a small RNA world. Curr Biol. 2001 Oct 2;11(19):R772–R775. [PubMed]
  • Paul Cynthia P, Good Paul D, Winer Ira, Engelke David R. Effective expression of small interfering RNA in human cells. Nat Biotechnol. 2002 May;20(5):505–508. [PubMed]
  • Yu Jenn-Yah, DeRuiter Stacy L, Turner David L. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):6047–6052. [PMC free article] [PubMed]

Articles from RNA are provided here courtesy of The RNA Society

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...