Wednesday, November 02, 2005
Your Data: Silencing Hepatitis C Virus Replication With In Vitro Transcribed siRNAs
The Silencer™ siRNA
Construction Kit uses an in vitro transcription reaction and
column purification to prepare transfection-ready siRNAs. With
this kit, multiple siRNAs can be prepared in ~24 hours. Below
is one example of how siRNAs prepared by the
Silencer siRNA Construction Kit were used
in gene silencing studies.
Kapadia S, Brideau-Anderson A, and
Chisari FV (2003) Interference of hepatitis C virus RNA
replication by short interfering RNAs. Proc Natl
Acad Sci USA 100: 2014-2018.
Dr. Sharookh B. Kapadia and colleagues at the Scripps
Research Institute used siRNAs prepared with the Silencer siRNA
Construction Kit to demonstrate that RNAi can be used to inhibit Hepatitis
C Virus (HCV) replication. The researchers generated multiple siRNAs targeting
various regions of the HCV subgenomic replicon sequence. These siRNAs
were transfected into an Huh-7 cell line that stably expressed the HCV
RNA replicon and screened for their gene silencing activities by real-time
RT-PCR. Two of the siRNAs, NS3-1948 and NS5B-6133, showed the greatest
specific inhibition of HCV RNA replication and were tested further for
their ability to suppress HCV RNA and protein expression. As shown in
Figure 1, HCV protein expression levels were significantly reduced on
Day 4 (Figure 1B) and Day 6 (Figure 1C) post-transfection. Similar results
are seen at the mRNA level (Figure 2). Viral replication was also inhibited.
These results suggest that RNAi might represent a new approach for the
treatment of HCV infection.
Figure 1. The
Effect of RNAi on HCV Protein Expression in S11791 Cells. Western blot analysis was performed
on total cell lysates harvested from mock- or siRNA-transfected S11791
cells at various days post-transfection by using NS3- and NS5B-specific
antibodies. Molecular mass markers (in kilodaltons) are shown to the right
of each gel. Shown are representative data of three independent experiments.
Figure 2. Effect of HCV-specific siRNAs on HCV
and GAPDH RNA Transcript Levels. Total RNA was isolated from S11791 cells
that were transfected with siRNA specific for NS3, NS5B, GAPDH, or an
irrelevant scrambled (scr) siRNA 2, 4, and 6 days previously. Equal amounts
of RNA were separated on a denaturing agarose gel, transferred to membrane
and hybridized with probes to HCV and GAPDH. GAPDH signal was used for
normalization of the HCV signal.
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Ordering Information
Cat#
Product Name
Size
1620
Silencer® siRNA Construction Kit
15 siRNA synthesis
rxns