RNAi Products

siIMPORTER siRNA TRANSFECTION AGENT

Millipore’s siIMPORTER siRNA Transfection Reagent sets a new standard for RNA Interference assays to cell signaling targets. This high efficiency, low cytotoxicity reagent is designed to work together with siRNA/siAb™ Assay Kits for maximum performance, convenience and value. When used together, these products provide an unbeatable pair for high transfection efficiency, low cytotoxicity and guaranteed siRNA-mediated gene knockdowns.

For studies using synthetic siRNA, siIMPORTER ensures high-quality, gene target specific knockdown data. The siIMPORTER siRNA Transfection Reagent works with a broad range of mammalian cells and may be used either in the presence or absence of serum in the culture medium. High transfection efficiencies ensure that the siRNA gets into a very large percentage of the transfected cell population while also providing low cytotoxicity.

siRNA/siAB ASSAY KITS

The family of siRNA/siAb™ Assay Kits is designed for convenience and ease-of-use. Kits include siRNA and antibodies to the same target, as well as positive and negative controls. The siRNA in each kit has been validated to knock-down the targeted mRNA by at least 75% in 24 hours, and the antibody has been validated to detect the targeted protein in Western blot assays. For more information products for RNA Interference, visit www.millipore.com/upstate.

siIMPORTER DELIVERY OF siRNA

AdenoSilence Library

The AdenoSilence library was designed and optimized specifically for RNA interference (RNAi) mediated gene-silencing. RNAi is a sequence specific post-transcriptional gene knockdown event that has become the standard format for studying gene function and elucidating drug target discovery. The viruses of the AdenoSilence library mediate RNAi through the intracellular expression of a short hairpin RNA (shRNA) that is driven by the strong and ubiquitous U6 promoter. The shRNA is then processed into small interfering RNA (siRNA), which is ultimately responsible for the degradation of target messenger RNA (mRNA) that contains complementary sequences. Every gene in the library is targeted by three separate viruses, each harboring a unique shRNA sequence that is specific to that gene. The sequence of each shRNA was selected by a proprietary search algorithm that identified putative shRNA sequences that contained no homology to other sequences within the mammalian genome. Numerous other selection criteria deemed vital for RNAi mediated gene-silencing were also met.

The AdenoSilence library is based on the adenovirus serotype 5 genome. The viruses are produced by a vector system in which two essential viral genes have been deleted and are provided by the proprietary packaging cell line, PerC6/E2A (Michiels et al., 2002). The flanking sequences of both genes were replaced such that no sequence overlap with the wild type genome exists. This sequence modification eliminates the possibility of homologous recombination, thus all of the viruses produced are replication incompetent. The viruses are capable of infecting cells but they cannot replicate after infection. Thus, the AdenoSilence viruses do not induce an infected cell to manufacture additional viruses and therefore they exert a very limited disruption to normal cellular metabolism.

Adenoviral infection is dependent upon the interaction between the capsid fiber variant and distinct cellular receptors. The AdenoSilence viral library was constructed using the C01 fiber variant which binds to the human coxsackievirus and adenovirus receptor (hCAR) protein (Bergelson et al., 1997). The hCAR protein is commonly expressed in numerous mammalian cell types, allowing the AdenoSilence viruses to infect an extensive number of different host cells, including primary cells. This broad tropism provides an important advantage for adenoviral based delivery systems over conventional transfection technologies (such as those used for plasmids or siRNA synthetics), which are hampered by toxicity issues and poor efficiencies in many different cell types, especially primary cells. Adenoviruses not only infect a wide variety of cell lines, they also typically display a high transduction efficiency in the cells they do infect. Additionally, the viruses mediate long term gene knockdown (often greater than 10 days), allowing efficient and extended silencing of the gene of interest (Arts et al., 2003). Unlike synthetic and plasmid based assays, which typically demonstrate shorter, more transient gene-silencing effects, the extended duration of gene knockdown provided by AdenoSilence viruses affords the researcher an opportunity to perform phenotypic assays that require longer term gene silencing such as cellular differentiation, cellular proliferation, and apoptosis analysis.

Adenoviruses also provide many other advantageous attributes. They are capable of infecting both dividing and non-dividing cells. This is in contrast to alternative viruses which can only infect actively dividing cells hence limiting their usefulness. Adenoviruses do not undergo chromosomal insertion of their genetic material into the host cells genome following infection. Therefore, their shRNA expression is not affected by the location of insertion. Finally, the adenoviruses consistently produce high titers of virus from the PerC6/E2A cell line. The high titers obtained aid in experimental setup since only a small volume of material is typically needed for testing.

Product Offering

Targeted Gene Classes

Druggable/Secreted
Enzymes
GPCRs
- poly(ADP-ribosyl)transferases
- methyltransferases
- choline acetyltransferases
- dehydrogenases
- deacetylases
- phospholipases
- proteases
Ion Channels
Kinases
Nuclear Hormone Receptors
Receptors
Transporters

pKD MAMMALIAN siRNA EXPRESSION PLASMIDS

siRNA – Mediated mRNA Degradation Pathway

Mammalian cells express endogenous, short genes using RNA polymerase III enzymes and promoters. Millipore’s Upstate plasmid-based siRNA constructs utilize this endogenous system to express short-hairpin RNA (shRNA) from pKD plasmids. The shRNA is ultimately processed by the cell to form a functional siRNA.

Advantages of pkD Mammalian siRNA Expression Plasmids

Pre-made and gene target-specific
Extensively validated in cell culture through either quantitative, real-time PCR or Western blotting
Expressed siRNA in vivo persists longer in the cell because as some is degraded, more is made to replace the loss
The plasmids can be stably integrated into the host genome
The sequence of the cloned siRNA is included at no additional charge
When more is needed, an inexpensive plasmid prep will generate more

Do you want a pKD for a gene target that we don't have?

Consider buying the empty vector version (ordering information below), and create your own gene target-specific pKD.

Extensive Validation of pKD Mammalian siRNA Expression Plasmids in Cell Culture

Millipore validates every plasmid in cell culture before release. Each plasmid is guaranteed to knock-down its intended target by at least 70% on the mRNA or protein level. The mRNA levels are determined using quantitative, real-time PCR, and protein levels are determined using Western blots.

Multiple Versions of Gene-Specific pKD siRNA Plasmids

Using multiple siRNAs to the same gene target is a useful control to validate specificity by confirming that a phenotype is the result of a targeted gene knock-down and not an off-target, non-specific effect. If the same phenotype results from multiple target-specific pKD siRNA plasmids, the likelihood that the phenotype is due to an off-target event is eliminated.

For most of Millipore pKD siRNA plasmids, multiple versions are available for use as specificity controls. Each version is specific for the same gene but is designed against different regions of the target’s mRNA.

pKD Series of Mammalian siRNA Expression Plasmids

Cloning into the pKD mammalian siRNA expression plasmid. In the pKD plasmid (cat. no.62-001), use the Smal site immediately downstream of the H1 promoter and any of the other sites to clone in the double stranded DNA oligos that will be expressed as a shRNA (short-hairpin RNA) which will get processed into a siRNA. By using the blunt Smal site, less non-specific sequence will be contained in the shRNA transcript. Make sure to include in the cloned DNA oligos a stretch of 5 thymidine residues that will serve as a transcription terminator.
All of the pKD siRNA plasmids have a similar structure. A human H1 promoter drives the expression of a cloned sequence that when transcribed forms a short-hairpin RNA (shRNA). The transcription is terminated by a dT sequence, and the shRNA gets processed by the cell into an siRNA. pKD (cat. no. 62-001) is available to clone your own double stranded DNA oligos into, or you can purchase any of the validated, gene target specific pKD-siRNA plasmids.

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