Synthetic Dicer-Substrate siRNAs as Triggers of RNA Interference - RNA Interference from Biology to Therapeutics - page 33

Fig. 2.1 Suppression of STAT1 expression. The ability of 21-nt siRNAs and 27-nt blunt dsRNA to
suppress STAT1 expression was studied at two sites. ( a ) STAT1 (NM_007315)-speci fi c dsRNAs were
transfected into HeLa cells at 1 nM or 0.1 nM concentration, and total RNA was isolated 24 h post-
transfection. RT-qPCR was performed on the STAT1 mRNA, and results were normalized to an inter-
nal HPRT1 control. ( b ) Sequences of the STAT1-specific dsRNAs employed are shown with the 27-nt
duplexes aligned under the 21-nt siRNA. RNA bases are uppercase and DNA bases are lowercase
but less dramatic increases in potency were observed at other sites within the EGFP
gene and also within a set of dsRNAs of varying length that targeted the Sjogren's
syndrome antigen B ( SSB ) gene and the heterogeneous nuclear ribonucleoprotein
H1 ( HNRNPH1 ) gene [ 30 ]. Thus, increased potency was observed using Dicer-
substrate siRNAs at multiple sites in three different genes in this study.
As the number of sites studied using this “first generation” blunt 27-nt design
was expanded, the situation become more complex. Inconsistent performance was
sometimes observed between sites such that some duplexes showed higher potency
in 27-nt blunt dsRNA form than in 21-nt siRNA form, others showed similar potency
between forms, while yet other sites showed higher potency in 21-nt siRNA form.
An example of this behavior is shown in Fig. 2.1 , where functional potency of