Confirming gene silencing,,,mechanism by pGFP/GFP22 ,,,siRNA co-transfection
In addition to the specificity and
efficiency of an siRNA sequence
for a target gene, which are of
high importance in post-transcriptional
gene silencing (PTGS), the
delivery of siRNA into cells is crucial.
An efficient method to optimize
transfection and gene silencing
is critical for successful RNAi
experiments. In this Application
Note HeLaS3 cells were transfected
with GFP expressing plasmid
and Cy5-labeled siRNA against
GFP, both individually and in
combination, using QIAGEN
transfection reagents. Uptake of
labeled siRNA and the gene
silencing effect were monitored
for up to 24 hours after transfection.
Efficient gene silencing was
achieved which confirms the
operation of RNAi mechanism in
the selected cell line.
The technique of RNAi has the
potential to revolutionize functional
genomics. The pathways and
mechanisms of RNAi are currently
under intense investigation, and a
good understanding of these is
essential to allow scientists to
exploit the applications of RNAi.
Simple and efficient methods for
monitoring gene silencing are
important tools for RNAi experiments.
In this Application Note we
describe the evaluation of a
method using fluorescent labels to
determine transfection efficiency
and the gene silencing effect.
HeLaS3 cells were transfected
with siRNA using RNAiFect
Reagent was used for the transfection
of GFP expressing plasmid (pGFP). Cells were also co-transfected
with both plasmid DNA and
siRNA using TransMessenger
Reagent. The uptake of siRNA into
mammalian cells was monitored
using fluorescent labeled siRNA
and the gene silencing effect and
level of cellular protein expression
were quantified at 4, 8, and 24
hours after transfection.
Page: All 1 2 3 4 Related biology technology :1
. Confirming RNAi Effects Is Now Easier Than Ever
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