MISSION esiRNA for RNAi screening in mammalian cells.
RNA interference (RNAi) is a basic cellular mechanism for the control of gene expression. RNAi is induced by short double-stranded RNAs also known as small interfering RNAs (siRNAs). The short double-stranded RNAs originate from longer double stranded precursors by the activity of Dicer, a protein of the RNase III family of endonucleases. The resulting fragments are components of the RNA-induced silencing complex (RISC), directing it to the cognate target mRNA. RISC cleaves the target mRNA thereby reducing the expression of the encoded protein(1,2,3). RNAi has become a powerful and widely used experimental method for loss of gene function studies in mammalian cells utilizing small interfering RNAs. Currently two main methods are available for the production of small interfering RNAs. One method involves chemical synthesis, whereas an alternative method employs endonucleolytic cleavage of target specific long double-stranded RNAs by RNase III in vitro. Thereby, a diverse pool of siRNA-like oligonucleotides is produced which is also known as endoribonuclease-prepared siRNA or esiRNA. A comparison of efficacy of chemically derived siRNAs and esiRNAs shows that both triggers are potent in target-gene silencing. Differences can, however, be seen when comparing specificity. Many single chemically synthesized siRNAs produce prominent off-target effects, whereas the complex mixture inherent in esiRNAs leads to a more specific knockdown(10). In this study, we present the design of genome-scale MISSION esiRNA libraries and its utilization for RNAi screening exemplified by a DNA-content screen for the identification of genes involved in cell cycle progression. We show how to optimize the transfection protocol and the assay for screening in high throughput. We also demonstrate how large data-sets can be evaluated statistically and present methods to validate primary hits. Finally, we give potential starting points for further functional characterizations of validated hits.
Theis
Mirko
M
Max Planck Institute of Molecular Cell Biology and Genetics.
Buchholz
Frank
F
D000818:Animals; D004247:DNA / Q000737:chemistry / Q000235:genetics; D015698:Genomic Library; D006801:Humans; D008322:Mammals; D034622:RNA Interference; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000235:genetics; D034741:RNA, Small Interfering / Q000138:chemical synthesis / Q000235:genetics*
D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D004247:DNA
J Biomol Screen. 1999;4(2):67-73
10838414
Proc Natl Acad Sci U S A. 2005 Feb 15;102(7):2396-401
15695330
Nat Methods. 2007 Apr;4(4):337-44
17351622
Nat Rev Genet. 2009 Feb;10(2):94-108
19148191
Cell Cycle. 2008 Jul 15;7(14):2123-8
18635956
Nat Biotechnol. 2006 Feb;24(2):167-75
16465162
Nat Biotechnol. 2003 Jun;21(6):635-7
12754523
Nat Methods. 2006 Oct;3(10):777-9
16990807
Nucleic Acids Res. 2004 Jul 1;32(Web Server issue):W113-20
15215362
Nat Cell Biol. 2007 Dec;9(12):1401-12
17994010