How do we achieve high potency & low off-target effect with SureSilencing
- HPLC Purification of Synthetic siRNA
Why HPLC purification matters?
The production of synthetic siRNA oligonucleotide strands begins at the 3'
end. Due to practical considerations of coupling efficiency, and depending on
the length and sequence of the target RNA, synthesis is often stopped before
full-length siRNA are generated. These truncated forms of siRNA (see figures
below) may result in sub-optimal performance, due to shifts
in thermostability, alterations
in specificity, changes to the
seed region and sequence-related
siRNA efficacy. Learn More
Purification of siRNA oligonucleotides by preparative high-performance liquid
chromatography (HPLC) is the most common, and most effective, method for
removing incompletely synthesized oligonucleotides and other contaminants. This
additional quality control step provides siRNA with >97% purity. Therefore,
HPLC-purified SureSilencing siRNA is ensured to provide high potency and low
off-target effect for knockdown of your specific gene.
High Purity of SureSilencing siRNA. SureSilencing siRNA and
siRNA from competitor X were analyzed side-by-side with 2100 Bioanalyzer. HPLC
purified SureSilencing siRNA (center lane) demonstrated over 97% purity with
right size, while siRNA from competitor X demonstrated multiple bands with shorter
length siRNAs (right lane). The left lane is the molecular weight marker.
SureSilencing siRNA provide about 90% success rate. HeLa cells were
transfected with 10nM of each siRNA targeting 24 transcription factors using
SureFECT Transfection Reagent. 48 hours after transfection, total RNA was
collected and real-time qPCR was performed to determine the target mRNA level.
For each gene target, the first two siRNA designs were selected. About 90% of
SureSilencing siRNA showed greater than 70% knockdown of target gene.
SureSilencing siRNA showed potent knockdown at very low concentration. HEK-293H cells were transfected with different concentration of siRNA for GAPDH using SureFECT Transfection Reagent. 48h after transfection, GAPDH activity level was determined. SureSilencing siRNA demonstrated matchless potency in effectively knocking down target gene even at 0.625 nM.
Note: The usually recommended siRNA concentration for efficient knockdown (greater than 70% at mRNA level) is 10 ~ 30 nM.
Long Lasting Effect
SureSilencing siRNA showed extended period of knockdown. HEK-293H
cells were co-transfected with turbo-RFP expression vector and either turbo-RFP
SureSilencing siRNA or negative control siRNA. Expression of turbo-RFP in
HEK-293H cells was monitored for 6 days (144 hours). The expression of turbo-RFP
is significantly down with SureSilencing siRNA (lower panel) since Day 1 till
Day 6 after transfection. In contrast, the expression of tubo-RFP wasn't
affected with control siRNA (top panel).
- Top-of-the-line Algorithm