Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference

Abstract

In the present study, the relationship between short interfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using 62 targets of four exogenous and two endogenous genes and three mammalian and Drosophila cells. We present the rules that may govern siRNA sequence preference and in accordance with which highly effective siRNAs essential for systematic mammalian functional genomics can be readily designed. These rules indicate that siRNAs which simultaneously satisfy all four of the following sequence conditions are capable of inducing highly effective gene silencing in mammalian cells: (i) A/U at the 5' end of the antisense strand; (ii) G/C at the 5' end of the sense strand; (iii) at least five A/U residues in the 5' terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nt in length. siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells. Essentially the same rules for siRNA sequence preference were found applicable to DNA-based RNAi in mammalian cells and in ovo RNAi using chick embryos. In contrast to mammalian and chick cells, little siRNA sequence preference could be detected in Drosophila in vivo RNAi.

Ube2s regulates Sox2 stability and mouse ES cell maintenance

“…Sox2 and Ube2s ORFs were inserted into vectors pCAG-3xHA and pCAG-3xFlag, respectively, for overexpression. Nineteen base pair (bp) gene-specific oligonucleotides for RNA interference were designed with the criteria defined by the work of Reynolds et al 59 and Ui-Tei et al 60 The RNAi oligonucleotides were cloned into pSuperpuro (Oligoengine, Seattle, WA, USA) between BglII and HindIII sites, and 19-bp short hairpin RNAs (shRNAs) with a 9-bp loop were expressed by the pSuperpuro plasmids. The two target sequences of the Ube2s shRNAs are 5′-GCTACTTCCTGACTAAAAT-3′ and 5′-GGAGGTCTGT TCCGTATGA-3′.…”

LacdiNAc (GalNAcβ1-4GlcNAc) Contributes to Self-Renewal of Mouse Embryonic Stem Cells by Regulating Leukemia Inhibitory Factor/STAT3 Signaling

“…We generated shRNA expression vectors targeting b4GalNAc-T3 or enhanced green fluorescent protein (EGFP) (negative control) by inserting the appropriate double-stranded DNAs between the BamHI and HindIII sites of pSilencer 3.1-H1 (Ambion, Austin, TX, www.ambion.com). The sequences used for RNAi were designed, as described previously [40], using ''siDirect (http://sidirect.jp/esd/modules/modsiperfect/)'' and are listed in supporting information Table 1. Transient transfection was performed as described previously [21].…”

“…By evaluating alkaline phosphatase activity in glycan-related gene knockdown mESCs, LacdiNAc, which is synthesized by b4GalNAc-T3 [35], was identified as a candidate cell surface glycan required for self-renewal of mESCs. We designed two constructs that expressed different shRNAs targeting b4GalNAc-T3 (b4GalNAc-T3-1 and b4GalNAc-T3-2), as described previously [40], and additionally constructs that expressed shRNAs targeting EGFP as negative controls. We describe mESCs that have been transfected with EGFP shRNA expression plasmids as ''control cells'' throughout this article.…”

Regulation of vimentin by SIP1 in human epithelial breast tumor cells

“…A SIP1-specific siRNA sequence was designed using selection criteria as described (Brummelkamp et al, 2002;Ui-Tei et al, 2004). A double PCR approach was used to create shRNA expression cassettes containing the H1 promoter and both the sense and antisense shRNA sequences with a loop sequence in between.…”