The siRNA Expression Vectors from MoBiTec are designed to induce siRNA mediated
gene suppression. Specifically, an siRNA coding sequence of 45-50 nucleotides is cloned into an RNA polymerase III expression cassette. After transfection, the siRNA sequence is expressed as fold-back stem-loop structure that is subsequently processed in the cell into functional siRNAs.
The MoBiTec siRNA Expression Vectors contain either the U6 or the H1 RNA polymerase III promoter, allowing optimal expression in a wide variety of cell types. Additionally, the MoBiTec siRNA Expression Vectors are available with GFP reporter genes to allow easy determination of vector transfection efficiency. All of the siRNA Expression Vectors contain the neomycin resistance gene for convenient selection of stable cell lines.

The MoBiTec siRTrans GeneDisruptor PCR Kits provide a fast, convenient, and effective way to produce siRNAs in vitro without the need for cloning. The kits rely on the PCR technology to enable the production of sequence specific siRNAs. The desired siRNA expression cassettes are generated by a one step PCR, and are then transfected into cells. Subsequently, the cassettes are transcribed to produce sense and anti-sense siRNAs, which anneal in situ to form the duplex siRNAs that are effective for gene silencing.
The following diagrams illustrate how the siRTrans GeneDisruptor PCR Kits work:

The siRTrans GeneDisruptor PCR Kits contain DNA templates with the U6 or the H1 RNA polymerase III promoters, allowing optimal expression in a wide variety of cell types. Additionally, these kits are available with a GFP reporter gene cassette to allow easy determination of PCR fragment’s transfection efficiency.

siRTrans siRNA Transfection Reagent is a unique cationic lipid formulation specifically designed for efficient delivery of siRNAs (small interfering RNAs) into a wide variety of cell types. siRTrans is easy to use, exhibits low cytotoxicity compared to competitor reagents, and works efficiently with both adherent and suspension cells. Table 1 shows some popular cells lines that have been successfully transfected with siRTrans.

siRTrans siRNA Transfection Reagent Titration Plates provide a convenient highthroughput format for optimizing transfection conditions and performing siRNA transfection in 96-well plates. All wells (except those in Column 12) contain a predetermined amount of the siRTrans siRNA transfection reagent, a cationic lipid based reagent that has been extensively screened in many mammalian cell lines in order to achieve:

• Efficient delivery of siRNA
  
• Functional gene silencing post siRNA delivery
  
• Compatibility with diverse growth conditions (with and without serum)
  
• Low cytotoxicity

When compared to other commercially available transfection reagents, siRTrans consistently offers superior transfection efficiencies and more effective gene silencing. siRTrans siRNA transfection reagent Titration Plates are provided in 3 related configurations:

The siRTrans siRNA Transfection Reagent Titration Plate contains serially diluted transfection reagent across the columns of the plate. Each well contains 11.7 - 0.15 µl of lipid diluted in serum-free media to a final volume of 25 µl. This format allows for the determination of the optimum amount of siRTrans reagent needed for maximal siRNA delivery. Additionally, optimal siRNA quantity may be determined by the addition of decreasing amounts of siRNA down the rows of the plate.

The siRTrans siRNA Transfection Reagent Standard Plate (High) contains the fixed amount of 3.5 µl of siRTrans siRNA Transfection Reagent + 21.5 µl of serum free medium in each well of the plate. This allows for rapid transfections in a 96-well format when a higher optimal volume of siRTrans has been established using the Titration Plate or from previous experiments or data.

The siRTrans siRNA Transfection Reagent Standard Plate (Low) contains the fixed amount of 0.7 µl of siRTrans siRNA Transfection Reagent + 24.3 µl of serum free medium in each well of the plate. This allows for rapid transfections in a 96-well format when a lower optimal volume of siRTrans has been established using the Titration Plate or from previous experiments or data.

Features and Advantages:

• Economical and easy to use
  
• Highest gene expression in diverse cell types
  
• Excellent enhancement with GeneTrans Transfection Reagent and GeneTrans II
  Transfection Reagent
  
• Very good improvement with other transfection reagents
  
• Effective for both transient and stable transfection
  
• Extended shelf life

General Product Description:

The GenePusher Reagents are a set of chemical cocktails developed for MoBiTec to increase the gene expression level obtained with synthetic non-viral DNA delivery systems such as GeneTrans Transfection Reagent and GeneTrans II Transfection Reagent. They also can be used with all other commercially available transfection reagents. Three unique formulations have been developed for different cell types (Table 1). They are very easy to use: simply add the appropriate GenePusher reagent to your culture medium 4 hours posttransfection. The level of gene expression can be enhanced by 2 to 12 fold (Table 3).
One vial of GenePusher reagent (1.5 ml), at a 100x concentration, is sufficient for 150 transfections using 1 ml transfection volume.
The application of the GenePusher Reagent is cell type dependent. Indeed, some cells respond greatly to the GenePusher Reagent (see Table 1 and 3) whereas others are not responsive (Table 2). Since the GenePusher Reagents are cocktails of specific chemicals, they could affect cell phenotype to a certain degree. Moreover, cellular toxicity may be increased with these reagents for certain cells and optimization may be required.

The Recombinant Human Dicer Enzyme Kit uses an ultra-active form of human recombinant dicer enzyme that can cleave more than 95% of dsRNA template into 22 bp siRNAs within 12 hours under optimized reaction conditions. This provides sufficient siRNAs in every single species to achieve effective gene silencing.

Components

• (4 x)* 100 µl Recombinant Dicer Enzyme
  
• (4 x)* 200 µl Dicer Reaction Buffer
  
• (4 x)* 25 µl 50 mM MgCl₂ Solution
  
• (4 x)* 50 µl 10 mM ATP
  
• (4 x)* 100 µl Dicer Stop Solution
  
• (4 x)* 1 ml Nuclease-free Water

dsRNA Removal siRNA Purification Column

The siRNA/dsRNA Purification Kit is used to remove salts and unincorporated nucleotides from siRNA generated by Dicer enzyme-mediated digestion reactions or dsRNA generated by in vitro transcription reactions. It is ideal for purifying siRNA generated with the dsRNA-Slicer rec. human Dicer enzyme kit or dsRNA generated with the SpeedScript T7 Transcription Kit. Each column is sufficient for purifying 0.5 µg – 1.0 µg of siRNA or dsRNA generated with the above mentioned kits.

Components

• 20 RNA Purification Columns
  
• 4 x 4 ml Hydration Buffer
  
  

The SpeedScript T7 Transcription Kit utilizes a novel technology to allow rapid synthesis of 10 to 50 times the amount of RNA produced by conventional in vitro transcription reactions. The secret behind the high yield is that each DNA template is copied hundreds of times. The SpeedScript T7 Transcription Kit is ideal for preparing double-stranded RNA for use with the Dicer siRNA Construction Kit. Each SpeedScript T7 Transcription Kit provides sufficient reagents for performing 20 transcription reactions.

Components

• 40 μl T7 Enzyme Mix
  
• 40 μl T7 Reaction Buffer
  
• 160 μl NTP Mix
  
• 20 μl DNase I
  
• 700 μl 2x Gel Loading Buffer
  
• 1 ml Nuclease-free Water
  
• 700 μl LiCl Precipitation Solution
  
• 10 μl at 1 μg/μl GFP Control Plasmid
  
• 40 μl at 1 μg/μl 5’ Control Primer
  
• 40 μl at 1 μg/μl 3’ Control Primer