siRNA Test Kit
Cat # N0080
£500 manual (pdf)
Luciferase-based siRNA Target Test Kit
is designed to provide a quantitative approach for evaluation of sequence
potential to serve as an efficient target for RNA interference (RNAi). Current
computer-based RNAi target searching algorithms are not perfect, leaving the
probability of selected sequence to be a good target from 30% to 60%. It is
therefore clear that the ability of computer-selected sequence to induce RNA
interference has to be confirmed experimentally. The siRNA Target Test Kit
allows for screening a large number of potential siRNA target sequences in
simple transient transfection experiments, measuring the reduction in reporter
(firefly Luciferase from Photinus pyralis) gene expression.
In most commercially available vectors and kits intended for the
same purpose, the gene silencing potential of a target sequence is tested on the
fused transcript bearing both reporter gene and gene of interest. In such
systems some unpredictable factors like chimeric mRNA folding and target
accessibility may affect the observation of RNAi. Also the distance between the
target site and reporter gene coding region along the mutual messenger RNA plays
a role in the reporter gene silencing: even if mRNA is cleaved at the point of
siRNA target, translation of the reporter gene can still go on till the RNA
degradation by nonspecific nucleases reaches the reporter gene coding region.
Such kind of effects may obscure the real efficiency of particular targets.
Unlike other kits, the siRNA Target Test Kit measures the
efficiency of target sequences per se. The short target sequences are
located at defined positions immediately upstream or downstream of the reporter
gene coding region. Independent evaluation of the reporter gene silencing for
two different positions of the same target permits to avoid or reduce the
influence of target accessibility factor and other factors related to RNA
conformation, thus making comparison between various targets more reliable.
Another advantage of the siRNA Target Test Kit is that it makes possible
evaluation of the siRNA target potential even in those cases when the gene of
interest is not available as full-size cDNA clone.
The siRNA Target Test Kit consists mainly of three
vectors: one effector plasmid (psiRNA) producing double-stranded siRNA, and two
reporter plasmids (psiTEST-target-LUC and psiTEST-LUC-target) expressing
Luciferase reporter gene fused to siRNA target sequence (Fig. 1). The system
design is based on the fact that the nucleotide sequence of siRNA is identical
to the sequence of corresponding siRNA target. Thus the same short synthetic DNA
fragment has to be cloned into all three plasmids, giving rise to siRNA coding
sequence in effector plasmid and to the siRNA target sequence in both reporter
Fig. 1 Construction of effector
and reporter plasmids and their mechanism of action.
siRNA expression from the effector plasmid psiRNA is driven by
dual promoter expression cassette bearing human U6 and H1 small nuclear RNA
promoters in opposite orientation to each other. Such dual promoter constructs
were shown to efficiently express double-stranded siRNA molecules (1-2) which
could be directly accepted by RNA Interference Searching Complex (RISC), the
multi-enzyme complex with RNase activity specifically digesting messenger RNA at
the siRNA target site (3). This permits to avoid the stage of dicer treatment
which is necessary for hairpin siRNA producing vectors to convert
single-stranded hairpin shRNA to double-stranded siRNA.
structure of the double-promoter siRNA expressing cassette is shown in Fig. 2.
siRNA coding sequence N1-N19 is placed between the U6 and H1 promoters. RNA
synthesis driven by the U6 promoter starts from the nucleotide N1 which has to
be G, and terminates at the stretch of 5 T invading the body of H1 promoter. As
a result, produced RNA has 2 or 3 uridines at its 3' terminus. Similarly, the
RNA produced from the H1 promoter starts with the nucleotide N'19, terminates at
5 T stretch at the beginning of the opposite U6 promoter, and also bears 2 or 3
uridines at its 3' terminus. Two halves of siRNA join together, forming the
functional double-stranded siRNA with protruding 3'-termini.
A mixture of siRNA-producing effector plasmid and one of the
target-bearing reporter plasmids has to be cotransfected transiently into
mammalian cells. Measuring the reporter gene silencing due to the effect of RNA
interference will give a quantitative evaluation of the siRNA target potential
Fig. 2 siRNA
expression from the dual promoter effector plasmid psiRNA.