High-throughput selection of effective RNAi probes for gene silencing.

RNA interference (RNAi) is a process of sequence-specific posttranscriptional gene silencing mediated by double-stranded RNA. RNAi has recently emerged as a powerful genetic tool to analyze gene function in mammalian cells. The power of this method is limited however, by the uncertainty in predicting the efficacy of small interfering RNAs (siRNAs) in silencing a gene. This has imposed serious limitations not only for small-scale but also for high-throughput RNAi screening initiatives in mammalian systems. We have developed a reliable and quantitative approach for the rapid and efficient identification of the most effective siRNA against any gene. The efficacy of siRNA sequences is monitored by their ability to reduce the expression of cognate target-reporter fusions with easily quantified readouts. Finally, using micro array-based cell transfections, we demonstrate an unlimited potential of this approach in high-throughput screens for identifying effective siRNA probes for silencing genes in mammalian systems. This approach is likely to have implications in the use of RNAi as a reverse genetic tool for analyzing mammalian gene function on a genome-wide scale.

[1]  M. Esteban,et al.  Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): Mechanism of action , 2000, Apoptosis.

[2]  P. I. Marcus.  Interferon Induction by Viruses , 1984 .

[3]  O. Donzé,et al.  RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. , 2002, Nucleic acids research.

[4]  Ali Ehsani,et al.  Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells , 2002, Nature Biotechnology.

[5]  A. Poustka,et al.  Systematic subcellular localization of novel proteins identified by large‐scale cDNA sequencing , 2000, EMBO reports.

[6]  W. Forrester,et al.  A DNA vector-based RNAi technology to suppress gene expression in mammalian cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[7]  F. Buchholz,et al.  Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[8]  M. Amarzguioui,et al.  Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor. , 2002, Nucleic acids research.

[9]  T. Rana,et al.  RNAi in human cells: basic structural and functional features of small interfering RNA. , 2002, Molecular cell.

[10]  D. Sabatini,et al.  Microarrays of cells expressing defined cDNAs , 2001, Nature.

[11]  T. Doering,et al.  Silence of the strands: RNA interference in eukaryotic pathogens. , 2003, Trends in microbiology.

[12]  Y. Dong,et al.  Systematic functional analysis of the Caenorhabditis elegans genome using RNAi , 2003, Nature.

[13]  W. Huttner,et al.  Tissue-specific RNA interference in postimplantation mouse embryos with endoribonuclease-prepared short interfering RNA , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Stacy L DeRuiter,et al.  RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Yang Shi,et al.  Mammalian RNAi for the masses. , 2003, Trends in genetics : TIG.

[16]  P. I. Marcus,et al.  Interferon induction by viruses. XIII. Detection and assay of interferon induction-suppressing particles. , 1985, Virology.

[17]  R. Agami.  RNAi and related mechanisms and their potential use for therapy. , 2002, Current opinion in chemical biology.

[18]  Michael T. McManus,et al.  Gene silencing using micro-RNA designed hairpins. , 2002, RNA.

[19]  T. Tuschl,et al.  RNA interference is mediated by 21- and 22-nucleotide RNAs. , 2001, Genes & development.

[20]  G. Hannon,et al.  Germline transmission of RNAi in mice , 2003, Nature Structural Biology.

[21]  B. Stillman,et al.  Human origin recognition complex large subunit is degraded by ubiquitin-mediated proteolysis after initiation of DNA replication. , 2002, Molecular cell.

[22]  K Weber,et al.  Identification of essential genes in cultured mammalian cells using small interfering RNAs. , 2001, Journal of cell science.

[23]  E. Harlow,et al.  Antibodies: A Laboratory Manual , 1988 .

[24]  H. Hohjoh.  RNA interference (RNAi) induction with various types of synthetic oligonucleotide duplexes in cultured human cells , 2002 .

[25]  D. Engelke,et al.  Effective expression of small interfering RNA in human cells , 2002, Nature Biotechnology.

[26]  P. Sharp,et al.  RNAi and double-strand RNA. , 1999, Genes & development.

[27]  Patrick J. Paddison,et al.  An epi-allelic series of p53 hypomorphs created by stable RNAi produces distinct tumor phenotypes in vivo , 2003, Nature Genetics.

[28]  M. Okabe,et al.  Small interfering RNA and gene silencing in transgenic mice and rats , 2002, FEBS letters.

[29]  F. Chisari,et al.  Interference of hepatitis C virus RNA replication by short interfering RNAs , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[30]  T. Tuschl,et al.  Analysis of gene function in somatic mammalian cells using small interfering RNAs. , 2002, Methods.

[31]  A. Caudy,et al.  Role for a bidentate ribonuclease in the initiation step of RNA interference , 2001 .

[32]  F. Ishikawa,et al.  Successful inactivation of endogenous Oct-3/4 and c-mos genes in mouse preimplantation embryos and oocytes using short interfering RNAs. , 2002, Biochemical and biophysical research communications.

[33]  R. Bernards,et al.  A System for Stable Expression of Short Interfering RNAs in Mammalian Cells , 2002, Science.

[34]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.