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RNAi Gene Silencing System

Gene silencing by RNA interference (RNAi) in eukaryotic cell cultures requires double-stranded molecules known as short interfering RNA (siRNA). QIAGEN is a licensed supplier of siRNA, offering a custom siRNA oligonucleotide synthesis service, library siRNA directed against common target genes, and a Cancer siRNA Oligo Set for screening a large number of cancer-related genes using RNAi techniques. Features and benefits
  • siRNA synthesis using patented TOM-Amidite chemistry for high-coupling efficiency
  • High-purity siRNA for efficient gene silencing
  • Expert advice on siRNA design for maximizing gene-silencing potential
  • Labeled siRNA allowing transfection to be easily followed
  • siRNA is annealed and ready-to-use in cell transfection
Principle The application of RNAi to mammalian cells has revolutionized the field of functional genomics by allowing targeted inhibition of gene expression using double-stranded RNA (dsRNA) that carries the same sequence as mRNA transcribed from the target gene. In mammalian cell systems, double-stranded siRNA oligos can be used to induce gene silencing.

After siRNAs are delivered to the cytoplasm of a cell, they bind to a multi-protein complex to form an RNA-induced silencing complex (RISC). The siRNA-protein complex targets en dogenous mRNA whose sequence matches that of the siRNA. The RISC nuclease activity then cleaves the mRNAsiRNA duplex resulting in silencing of the target gene (see flowchart "siRNA-Mediated RNAi").

To target a specific gene by RNAi, a double-stranded siRNA containing the sense and antisense sequence of mRNA transcribed from the gene is required. QIAGEN provides expert advice on siRNA design. Patented TOM-Amidites are used to synthesize high-quality, high-purity, 21-nucleotide sense and antisense RNA oligonucleotides that are annealed to produce a duplex with symmetric 2-nucleotide 3'-end overhangs. Deprotected siRNA duplexes are desalted and purified to >97% purity by HPLC (ultrapure) or 80-85% (crude option). siRNA is supplied with a sterile resuspension buffer and is ready to use in transfection procedures.

Efficient Gene Silencing Using QIAGEN siRNA and TransMessenger Reagent Western blot of extracts from transfected HeLa-S3 cells probed with lamin A/C monoclonal primary antibodies. Cells were transfected using the indicated reagents. siRNA: short interfering RNA duplex targeting lamin A/C at position 608630 relative to the start codon; TM: TransMessenger Transfection Reagent; ODN: Scrambled 24mer oligodeoxynucleotide (control).

Applications
  • Repress expression of endogenous genes
  • Study of molecular and cellular processes
  • Target validation studies
  • Potential for gene therapy
Efficient, Targeted Gene Silencing Relative expression levels of lamin A/C after siRNA transfection. The value for the mock transfection was set as 1. Data were normalized to the expression level of GAPDH (internal control), which was quantified by real-time RT-PCR using Omniscript Reverse Transcriptase and the QuantiTect SYBR Green PCR Kit. Expression levels were calculated using a standard curve generated using different amounts of HeLa S3 cDNA. Data obtained under the indicated transfection conditions are shown as relative, normalized expression levels. Total RNA was prepared using the RNeasy Mini Kit and the QIAShredder homogenizer.



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