TransIT-TKO HTS-96 Plates

Features

• Ideal for screening applications: High efficiency knockdown in a 96-well plate format allows rapid, simultaneous screening of multiple siRNA conditions including siRNA concentration, sequence and cell type in one plate.

• Customised: Plates are available in four working concentrations of TransIT-TKO® Reagent for optimisation with a wide range of cell types. The Titration Plate is available for testing all four levels at one time in a preliminary screen.

• Convenient: Easy to use, serum compatible protocol; can be performed in a single 96-well plate or using a two step transfer method.

• Consistent performance: Efficient and reproducible siRNA delivery among multiple wells in a plate for performing replicate samples.

DESCRIPTION

TransIT-TKO® Transfection Reagent enables highly efficient siRNA transfection with significantly reduced levels of cell damage as compared to cationic-liposome based transfection reagents. Transfections are most effective when carried out in complete growth media, with no media change or serum addition required. TransIT-TKO® Reagent, when complexed with siRNA, knocks down target gene expression in a variety of cell lines.

The TransIT-TKO® HTS-96 Plates were developped by Mirus Corporation to accelerate and simplify methods for high throughput screening in gene silencing applications. The TransIT-TKO® Reagent is supplied as a dry film on clear plastic, sterile, 96-well plates which have flat-bottom wells suitable for cell culture. Each plate is supplied individually vacuum sealed in a foil pouch to maintain sterility and ensure effectiveness. Researchers can test up to eight different siRNA sequences in triplicate format, at four different levels of reagent, from L1 (lowest) to L4 (highest), using the TransIT-TKO® HTS-96 Titration Plate (AM7180), on their particular cell type. Once the optimal level has been determined based on the highest efficiency of transfection with the lowest cellular toxicity, additional plates can be purchased at the desired level. Transfections can be performed using a single TransIT-TKO® HTS-96 Plate or using a two step transfer method, where the TransIT-TKO® HTS-96 Plate is used as a reagent plate to form complexes and transfer to the plate of choice.

Cell Lines Successfully Tested:

A549, BNL.CL2, HeLa, Hepa1CLC7, HepG2, Neuro-2a, NIH3T3. Stably integrated cell lines: 293-lux, 3T3-lux, CHO-luc, CHO-SEAP.

RNA Interference Overview:

Cellular uptake of long double stranded RNA (dsRNA) has been shown to induce RNA interference in a diverse group of organisms as well as insect cells in culture. RNA interference leads to the inhibition of protein expression by utilising sequence-specific, dsRNA-mediated destruction of the target messenger RNA (mRNA). Attempts to induce RNA interference using long dsRNA in mammalian cell lines has been met with limited success, due in part to the induction of the interferon response, which results in a general inhibition of protein synthesis. Recently, it has been shown that when short RNA duplexes are introduced into mammalian cells in culture, sequence-specific inhibition of target mRNA can be realised without introducing an interferon response. These short dsRNA, referred to as small interfering RNAs (siRNA), act at sub-molar ratios to cleave greater than 95% of the target mRNA in the cell. The RNA interference effect can be long-lasting and may be detectable after many cell divisions. These properties make siRNA extremely effective at inhibiting target gene expression once introduced into the cell.

The data below demonstrate the use of the TransIT-TKO® HTS-96 Titration Plate to determine the optimal level of TransIT-TKO® Transfection Reagent for achieving high efficiency knockdown while maintaining optimal cell viability in specific cell lines.

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