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Gene Knockdown

Integrated Solutions for Gene Knockdown

Introduction
The RNA interference (RNAi) pathway was first discovered in Caenorhabditis elegans as a response to double-stranded RNA (dsRNA) resulting in sequencespecific gene silencing. Due to its exceptional specificity and efficiency, RNAi has become a powerful gene knockdown technique widely used for the analysis of gene function as well as for target identification and target validation. A prerequisite for successful gene knockdown is the efficient transfection of small interfering RNA (siRNA). With X-tremeGENE siRNA Transfection Reagent, Roche Applied Science has introduced a new high-quality reagent for effective gene knockdown in siRNA- and cotransfection-based experiments. To ensure that the resulting cellular effects are specific for silencing the target gene, siRNA action can be monitored directly using the LightCycler System for relative quantification of the target mRNA. The real-time PCR data help you identify functional siRNA sequences that mediate efficient silencing.

For knockdown verification on the protein level, Western blotting is the method of choice. Using the sensitive LumiLightPLUS Western Blotting Substrate saves precious sample material. The Roche Applied Science portfolio is completed by a variety of assays for the functional analysis of downstream effects associated with cell growth and cell death (e.g., the Cell Proliferation Reagent WST-1). We at Roche Applied Science utilize our experience to meet your needs in RNAi research and to provide reliable tools facilitating the different steps of your gene knockdown experiment: siRNA generation, transfection, knockdown verification, and functional analysis. Major products for gene knockdown are described in more detail here.

siRNA Generation
Plasmid purification When plasmids expressing short hairpin RNA (shRNA) are used for RNAi research, the purity of the plasmid DNA is crucial for a successful transfection step. Plasmid purification with Genopure Plasmid Kits yields highly pure, transfection-grade plasmid DNA. For more information, visit www.roche-applied-science.com/napure

Tools for Gene Knockdown

siRNA Generation

Transfection

Knockdown Verification

Functional Assays

Plasmid Purification

siRNA Transfection

RNA Isolation

Protein Stabilization

Cell Proliferation Assays

Plasmid Transfection

Real-Time PCR

Western Blotting

Apoptosis Assays

Northern Blotting

Epitope Tagging

Cytotoxicity Assays

Reporter Gene Assays

Parameter-Specific ELISAs

Figure 1: Roche Applied Sciences product portfolio for gene knockdown applications

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Gene Knockdown Transfection

siRNA transfection X-tremeGENE siRNA Transfection Reagent efficiently delivers siRNA into many commonly used cell types including HeLa, NIH3T3, HEK293, CHO-K1, COS-7, and several hard-to-transfect cell lines such as HT29 (a human adenocarcinoma cell line). When used for cotransfection-based gene knockdown, X-tremeGENE siRNA Transfection Reagent enables significant protein expression and effective knockdown levels due to its high transfection efficiency. Since X-tremeGENE siRNA Transfection Reagent functions exceptionally well in the presence or the absence of serum and demonstrates low cytotoxicity, it eliminates the need to change media. Plasmid transfection We recommend FuGENE 6 Transfection Reagent for the transfection of plasmids expressing shRNA. FuGENE 6 Reagent is an advanced nonliposomal formulation enabling transfection of a wide variety of eukaryotic cell types (more than 700) including primary cultures and hard-to-transfect cell lines with high efficiency and minimal cytotoxicity. For more information, visit www.roche-applied-science.com/transfection

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Knockdown Verification
Gene knockdown can be measured on the mRNA and/or on the protein level. Roche Applied Science offers a variety of systems and reagents for reliable knockdown verification, both on mRNA and protein level. yDetection on mRNA level RNA isolation The RNA isolation step can be carried out either manually or automated: Roche Applied Science offers kits for the manual isolation of RNA, e.g. High Pure RNA Isolation Kit and mRNA Isolation Kit. The MagNA Pure LC System allows the fully automated isolation of total RNA or mRNA from up to 32 samples in parallel. For more information, visit www.roche-applied-science.com/napure or www.magnapure.com Real-time PCR Roche Applied Science LightCycler Technology set the standard for rapid, sensitive, and accurate real-time PCR. Using the LightCycler 2.0 Instrument, you can monitor the amplification of the PCR product simultaneously, in realtime and online, with six different detection channels. Analysis of the results of gene knockdown studies is substantially improved with our new relative quantification software and solutions for multiplex PCR, leading to novel approaches in assay design. The LightCycler System

comprises instruments, software, and reagents. For more information, visit www.roche-applied-science.com/lightcycler Northern blotting Quantify RNA using nonradioactive labeling methods: Nonradioactive labeling systems such as the DIG system from Roche Applied Science have become increasingly popular. They provide the same degree of sensitivity and are much faster, and more convenient to use than radioactive labeling systems. Benefit from the advantages of the DIG system using the DIG RNA Labeling Kit (SP6/T7) in combination with the DIG Luminescent Detection Kit for Northern blotting. For more information, visit www.roche-applied-science.com/dig yDetection on protein level Protein stabilization Cells have different types of proteases; mixtures of different inhibitors are needed to completely protect proteins for subsequent experiments (e.g., specific protein-activity assays). Our C mplete Protease Inhibitor Cocktail Tablets contain a specially optimized mixture of different protease inhibitors. The addition of one tablet to the sample

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Gene Knockdown

assures convenient, reliable protection of the protein of interest. For more information, visit www.roche-applied-science.com/proteaseinhibitor Western blotting Detect protein by colorimetric or chemiluminescence methods: the detection of proteins using chemiluminescence has become the method of choice, with peroxidase and luminol reagents being the most popular chemiluminescence system for protein detection. You can save sample material by using the sensitive Lumi-LightPLUS Western Blotting Substrates, Kits, and polyvinylidene difluoride (PVDF) membranes. The Lumi-LightPLUS substrate enables multiple exposures due to the high stability of the signal after substrate addition (stable for more than nine hours). For more information, visit www.proteomic-science.com Epitope tagging Epitope tagging eliminates the laborious and time-consuming task of producing a new antibody every time a protein is studied. Our anti-Tag antibodies simplify the detection, characterization, and purification of proteins. They are also available in conjugated forms, and recognize commonly used epitope sequences, such as the influenza hemagglutinin protein (HA), human c-myc protein, and six histidine residues (His6). For more information, visit www.proteomic-science.com Reporter gene assays Roche Applied Science offers a broad line of sensitive kits and reagent sets for reporter gene assays on transfected cells. These nonradioactive products with a sensitivity equal to or greater than that of radioisotopic assays, provide a safe alternative for accurate results. For more information, visit www.proteomic-science.com

diseases. For the study of apoptosis-related targets, Roche Applied Science provides various tools (e.g., the Cell Death Detection ELISAPLUS measuring DNA fragmentation) to fulfill the demands of such screening studies. For more information, visit www.roche-applied-science.com/apoptosis Cytotoxicity assays Most current assays for measuring cytotoxicity are based on alterations in plasma membrane permeability and the subsequent release (leakage) of components into the supernatant, or the uptake of dyes, which are normally excluded by viable cells. The Cytotoxicity Detection Kit (LDH) is a precise, fast, and simple colorimetric assay for the quantitation of cytotoxicity/cytolysis based on the measurement of lactate dehydrogenase (LDH) activity released from damaged cells. Thus, the Cytotoxicity Detection Kit (LDH) can be used in many different in vitro cell systems to determine plasma-membrane damage. For more information, visit www.roche-applied-science.com/apoptosis Parameter-specific ELISAs An enzyme-linked immunosorbent assay (ELISA) for biomedical applications must be carefully developed and excellently manufactured. ELISAs from Roche Applied Science are designed to be quick and accurate. One-step immunoassays save time and result in less error. Special additives eliminate interference from human anti-mouse antibodies (HAMA). We offer ELISAs for various fields in drug and disease research, such as cytokine and growthhormone research, and oncology research. Moreover, streptavidin-coated microplates (Strepta-Well) with high binding capacity for chemiluminescence, colorimetric, or fluorescence detection are available. For more information, visit www.proteomic-science.com

Functional Assays
Cell proliferation assays Cell proliferation and viability assays are of particular importance for routine applications in RNAi research. Tetrazolium salts (e.g., WST-1) are especially useful for this type of analysis. Cell Proliferation Reagent WST-1 yields water-soluble cleavage products, which can be measured without an additional solubilization step to determine cell viability e.g., after transfection with siRNA. For more information, visit www.roche-applied-science.com/apoptosis Apoptosis assays Apoptosis- and cell proliferation-related genes and proteins have become quite relevant putative drug or diagnostic targets since many studies have demonstrated that deregulation of cell death is associated with a variety of

New Internet Site

For more information on our complete product portfolio for gene knockdown applications visit www.roche-applied-science.com/geneknockdown (coming soon)

Product X-tremeGENE siRNA Transfection Reagent

Pack Size

Cat. No.

1 ml 04 476 093 001 (400 transfections in a 24-well plate) Multi-pack 04 476 115 001 5 x 1 ml (2,000 transfections)

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