Long-Term Gene Suppression

Short, double-stranded RNA molecules (dsRNA) have traditionally been synthesized in vitro, making them very expensive to produce. A new technique using a pol III promoter to express a short-hairpin RNA (shRNA) molecule in vivo has become very effective to reduce gene expression in a more economical fashion. The GeneEraser^® shRNA expression system* consists of the pGE-1 vector and appropriate positive and negative controls for setting up the system in your lab (Figure 1). We have chosen the human U6 promoter for its effective expression of shRNA. This promoter has consistently shown the highest suppression of the luciferase control in a variety of cell lines, consistently reducing expression by 80 to 95% (data not shown). This vector is available either supercoiled or as a predigested cloning kit.

Fast, Economical siRNA Production

Recombinant Dicer Enzyme** is the most costeffective and rapid method available for generating small interfering RNA (siRNA). This enzyme eliminates guesswork and the time-consuming and expensive design/synthesis of multiple siRNAs for screening. This enzyme digests a long targetspecific dsRNA molecule into short siRNAs. This creates a comprehensive pool of siRNA molecules that represent the entire target region. These siRNAs are then transfected into mammalian cells. After transfection into cells, we have seen up to 95% suppression of the target gene. We have simplified the protocol by eliminating the unnecessary ATP cofactors that are required for native Dicer enzyme (Figure 2). Use with GeneEraser™ siRNA transfection reagent*** for optimal results.

Optimized siRNA Transfection

RNA interference experiments using siRNA require a transfection reagent that has low toxicity and is specially optimized for siRNA delivery. This lowtoxicity reagent significantly reduces cell damage compared to cationic-liposome-based transfection reagents and outperforms these reagents in silencing experiments (Figure 3). Very low levels of siRNA are required for transfections with this reagent to knock out stable or transient gene expression in a wide variety of cell lines. This optimized protocol allows you to transfect in the complete growth media, eliminating the need for media changes and the addition of serum.

Easily Produce Large Quantities of RNA

The RNAMaxx™ high yield transcription kit is designed to produce large quantities of RNA quickly and easily from a DNA template. Just 1 µg of template can yield 80 to 100 µg or more of RNA transcript in one short incubation step. This is 10 to 30 times the yield produced by a traditional in vitro transcription reaction. The kit uses T7 RNA polymerase to produce high yields of RNA in just two hours. RNAMaxx high yield transcription kit outperforms competitor’s enzymes (see page 10).

Figure 3
Silencing Effect of Luciferase siRNA
83 to 90% knockout of luciferase expression was observed when 2 to 30 nM concentration of siRNA were used to complex with the GeneEraser reagent. Only 40 to 60% knockout was observed with Reagent A and only 13 to 22% with Reagent B. For the transient luciferase expression, HeLa cells were transfected with 10 µg luciferase reporter plasmid pGL2 (Promega) in T75 flask using Stratagene’s GeneJammer^® transfection reagent. Two hours later, cells were split into a 24-well plate, allowed to adhere for an additional two hours, and then transfected with luciferase GL2 siRNA duplex (Dharmacon Research) using different transfection
reagents (2.5 µl). Twenty-four hours post-transfection, cell lysates were assayed for luciferase activity using Stratagene’s Luciferase Assay Kit.

See license reference 10
** See license reference 11
*** See license reference 12

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