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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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