Target Discovery and Validation Reviews and Protocols

RNAi Libraries 139
Fig. 4. Example of analysis of dsRNA-induced apoptosis by using siRNA expression
vector library. HeLa S3 cells were transfected with siRNA expression vectors directed
against the gene for PKR at two different sites (site 1 and site 2) or with negative control
vectors (NC1 and NC2). After selection with puromycin, the cells were transfected
with poly(I:C). (A) Western blotting analysis of extracts of cells transfected with siRNA
expression vectors directed against the gene for PKR (site 1 and site 2; PC1 and PC2)
and negative controls (NC1 and NC2). (B) Plates after staining with crystal violet.
(C) Microscopic images of cells. (D) The wells surrounded by the circle (S1–S4) contain
cells that survived under the induction of apoptosis.
region of siRNA sequences. Introducing mutations only into the sense strand of
the hairpin RNAs could reduce these problems. Loop sequence derived from natural
microRNA sequence was found to improve silencing efficacy.
3. The followings are the guidelines for choosing the gene targets: avoid stop
sequences of pol III (UUUU and UUAUU), regions with GC content 60%, repeat sequences, and sites containing single nucleotide polymorphisms.
However, the siRNA should have low internal stability at the 5′-end antisense
strand, high internal stability at the 5′ sense strand (but if there is low internal stability
at the 5′ end antisense strand, this criterion is not so critical), presence of A
(position 19) and U (positions 10 and 19) of the sense strand, absence of G or C
(position 19) of the sense strand and absence of close homology to other gene
sequences.
4. This protocol was designed for HeLa S3. When other cell lines are used, the following
conditions need to be optimized: type of transfection reagents, amount of
DNA, used for transfection optimal cell density before transfection and drug