Stealth RNAi Collections

Stealth RNAi Collections Catalog nos. 12938-200, 12938-400, 12938-500, 12938-600, 12938-700, 12938-800, 12940-600 Version E 22 March 2006 25-0878 User Manual

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Table of Contents Table of Contents... iii Contents and Storage... iv Accessory Products... v Introduction... 1 Overview...1 Plate Layout and Control Information...5 CD-ROM Information...9 Experimental Overview...11 Methods... 12 General Guidelines...12 Reverse Transfection Protocol...18 Detecting Control Signal...25 Data Analysis Guidelines...26 Reordering Stealth RNAi...28 Troubleshooting...30 Appendix... 32 Technical Service...32 Purchaser Notification...33 References...35 iii

Contents and Storage Products This manual is shipped with the following products: Product Catalog no. Stealth RNAi Human Kinase Collection 12938-200 Stealth RNAi Human Phosphatase Collection 12938-400 Stealth RNAi Human Nuclear Receptor Collection 12938-500 Stealth RNAi Human Ion Channel Collection 12938-700 Stealth RNAi Human Non-Olfactory GPCR Collection 12938-800 Stealth RNAi Mouse Nuclear Receptor Collection 12938-600 Stealth RNAi Mouse Ion Channel Collection 12940-600 Shipping and Storage Contents The Stealth RNAi Collection is shipped on dry ice. Upon receipt, store at -20 C. Products are guaranteed for one year from date of shipment when stored properly. Each Stealth RNAi Collection is comprised of a set of 96-well plates and a CD-ROM (includes detailed plate and gene information, see page 9). Two nmoles of each Stealth RNAi is supplied in 100 µl 1X RNA Annealing/Dilution Buffer (10 mm Tris-HCl, ph 8.0; 20 mm NaCl; 1 mm EDTA, ph 8.0) at a final concentration of 20 µm. Quality Control The Stealth RNAi Collection is qualified as follows: The identity and concentration of each corresponding single-stranded RNA oligo is verified by mass spectrometry and optical density reading, respectively. After annealing, the Stealth RNAi duplex is analyzed by non-denaturing polyacrylamide gel electrophoresis to confirm duplex formation and the absence of RNA degradation. The Stealth RNAi duplexes and appropriate controls are arrayed in 96-well plates using a validated, automated aliquoting process. Each well must contain a minimum of 2 nmoles of Stealth RNAi in solution at a final concentration of 20-27 µm. iv

Accessory Products Additional Products Additional products that may be used with the Stealth RNAi Collection are available from Invitrogen. Ordering information is provided below. Item Quantity Catalog no. Lipofectamine 2000 Reagent 0.75 ml 1.5 ml 11668-027 11668-019 Lipofectamine RNAiMAX Reagent 0.75 ml 1.5 ml 13778-075 13778-150 Opti-MEM I Reduced Serum Medium 100 ml 31985-062 500 ml 31985-070 BLOCK-iT Transfection Kit 1 kit 13750-070 Stealth RNAi Negative Control Kit 1 kit 12935-100 BLOCK-iT Fluorescent Oligo 2 x 125 µl 2013 75 µl 13750-062 mrna Catcher PLUS 1 x 96-well plate 10 x 96-well plates K1570-02 K1570-03 CyQUANT Cell Proliferation Assay Kit 1 kit C7026 v

Accessory Products, Continued CellSensor Cell Lines and Antibodies CellSensor Cell Lines use the GeneBLAzer Technology to provide you with a reliable, rapid, and sensitive method of analyzing the intracellular status of signal transduction pathways upon exposure to drug candidates or other stimuli. Each CellSensor Cell Line stably expresses a response element coupled to the beta-lactamase reporter. When pathways leading to the response element are activated or inhibited, beta-lactamase reporter activity is modulated and is measured with the GeneBLAzer Loading Substrates. A large variety of high-quality antibodies including the Zymed Antibodies is available from Invitrogen for use in immunohistochemistry or ELISA assays. For details, visit www.invitrogen.com or contact Technical Service (page 32). KIF11 Stealth Select RNAi To qualitatively assess transfection efficiency for Lipofectamine RNAiMAX transfections, we recommend using a KIF11 Stealth Select RNAi. To order, go to www.invitrogen.com/rnaiexpress and search for gene symbol KIF11, or contact Technical Service (page 32). For human cells, we recommend oligo HSS105842; for mouse cells, oligo MSS205751. vi

Introduction Overview Introduction The Stealth RNAi Collection combines the power of chemical modification with advanced in silico design algorithms to provide you with an advanced RNAi collection suited for RNA interference (RNAi) studies. The collection is composed of three, non-overlapping Stealth RNAi duplexes designed to target human or mouse genes that are arrayed in 96-well format for high-throughput gene silencing screening experiments. Stealth RNAi Collection The Stealth RNAi Collection is comprised of Validated Stealth RNAi duplexes and/or Stealth Select RNAi duplexes (see CD-ROM for details) that target human or mouse targets arrayed in 96-well plates. Each Stealth RNAi duplex is supplied at 2 nmoles per well which is sufficient to perform ~200 transfections. Additional positive and negative controls as well as blank wells for user designed controls are included on each plate. See page 3 for details on Validated Stealth RNAi and Stealth Select RNAi. Target Genes The Stealth RNAi Collection is composed of three, nonoverlapping Stealth RNAi duplexes designed to target human or mouse genes. The target genes represent a portion of the druggable genome and include genes from important gene families including protein kinases, phosphatases, and nuclear receptors that are increasingly attractive targets for drug screening and therapeutic development. 1

Overview, Continued Advantages of Stealth RNAi Collection Using the Stealth RNAi Collection provides the following advantages: Available as a collection of Stealth Select and Validated Stealth RNAi duplexes Allows guaranteed screening success with higher target specificity, reduced off target effects, and high quality results. Includes appropriate positive, negative, and transfection controls Enables transfection efficiency evaluation and proper analysis of the data to allow identification of significant hits. Collection arrayed in a easy-to-use 96-well format that is compatible for use with manual or automated screening System Overview The Stealth RNAi Collection is arrayed in a 96-well format to allow high-throughput screening. To use the collection, transfect cells with the Stealth RNAi and allow for protein turnover. Perform colorimetric, fluorescent, or luminescent cell-based screening assays in a 96-well format to determine gene function, elucidate gene pathway, or validate therapeutic targets. If you have access to sophisticated imaging devices and algorithms, you can use microscopy-based, high-content fluorescent screening assays to determine the relative fluorescence and the subcellular localization of the target gene. The screening allows you to identify genes ( hits ) for which the Stealth RNAi produces a specific phenotype. Accessing Certificate of Analysis (C of A) To access the Certificate of Analysis (C of A), go to www.invitrogen.com/cofa. Input the lot number found on the Box label of the Stealth RNAi Collection or on the packing slip supplied with the product. Note that you will download a separate C of A for each box of the collection. You will be unable to access the C of A if you input the lot number located on each plate of the collection. You must use the lot number located on the box label. 2

Overview, Continued Stealth RNAi Stealth RNAi is chemically modified, blunt end dsrna developed to overcome the limitations of traditional sirna. The two strands of Stealth RNAi are modified in a manner that prevents sense strand activity, eliminating sense strand mediated-off-target activity. The Stealth RNAi interacts with the RNAi machinery (see next page) to elicit gene silencing, similar to traditional sirna. See next page for RNAi pathway. Using Stealth RNAi for RNAi analysis offers the following advantages: Obtain effective target gene knockdown at levels that are equivalent to or greater than those achieved with traditional sirna Reduces non-specific effects caused by induction of cellular stress response pathways Exhibits enhanced stability for greater flexibility in RNAi analysis The Stealth RNAi Collection is comprised of Validated Stealth RNAi (see below) and Stealth Select RNAi (see below) duplexes. Validated Stealth RNAi The Validated Stealth RNAi are functionally tested Stealth RNAi molecules designed for in vitro or in vivo RNAi analysis of a specific target gene. Each Validated Stealth RNAi duplex targets a different region of the gene of interest, demonstrating >80% target gene knockdown as measured in a cell-based assay. Stealth Select RNAi Stealth Select RNAi are pre-selected molecules designed using a state-of-art design algorithm that delivers highly specific and >70% knockdown of target gene. Information on the CD-ROM Detailed information on each Stealth RNAi including the gene ID (accession number), gene symbol, gene name, and location of each Stealth RNAi on the plate is included in the CD-ROM shipped with the kit. The RNAi sequence information is not included on the CD-ROM. To request RNAi sequence information, contact Technical Service (page 32). 3

Overview, Continued RNAi Pathway RNAi describes the phenomenon by which dsrna induces potent and specific inhibition of eukaryotic gene expression via the degradation of complementary messenger RNA (mrna), and is functionally similar to the processes of posttranscriptional gene silencing (PTGS) or cosuppression in plants (Cogoni et al., 1994; Napoli et al., 1990; Smith et al., 1990; van der Krol et al., 1990) and quelling in fungi (Cogoni & Macino, 1997; Cogoni & Macino, 1999; Romano & Macino, 1992). In plants, the PTGS response is thought to occur as a natural defense against viral infection or transposon insertion (Anandalakshmi et al., 1998; Jones et al., 1998; Li & Ding, 2001; Voinnet et al., 1999). In eukaryotic organisms, dsrna produced in vivo or introduced by pathogens is processed into 21-23 nucleotide double-stranded short interfering RNA duplexes (sirna) by an enzyme called Dicer, a member of the RNase III family of double-stranded RNA-specific endonucleases (Bernstein et al., 2001; Ketting et al., 2001). The sirna is then incorporated into an RNA-induced silencing complex (RISC), an enzyme complex that serves to target cellular transcripts complementary to the sirna for specific cleavage and degradation (Hammond et al., 2000; Nykanen et al., 2001). In addition to dsrna, other endogenous RNA molecules including short temporal RNA (strna) and micro RNA (mirna) (Ambros, 2001; Carrington & Ambros, 2003) have been identified and shown to be capable of triggering gene silencing. For more information about the RNAi pathway, refer to recent reviews (Bosher & Labouesse, 2000; Dykxhoorn et al., 2003; Hannon, 2002; Plasterk & Ketting, 2000; Zamore, 2001). Purpose of the Manual This manual provides the following information: Overview of the Stealth RNAi Collection Plate Layout and content information General guidelines for using the collection Instructions to perform the reverse transfection procedure Guidelines for data analysis Troubleshooting Protocols for screening assays are not included in this manual. 4

Plate Layout and Control Information Introduction General plate layout and information on the Stealth RNA controls included on the plate are described in this section. For gene information and location of each Stealth RNAi on the each plate, refer to the information included in the CD. Plate Specifications The Stealth RNAi Collection Plate specifications are listed below: Dimensions: Standard SBS (Society for Biomolecules Screening) footprint Description: Polypropylene 96-well plate with V-well bottom and a removable lid Working Volume/Well: 190 µl The plate is compatible for use with a multichannel pipettor, or 8-, 12-, or 96-tip robotic loading devices with an automated liquid handling workstation. The plate and lid have alphanumeric markings for easy well identification. Plate Layout The Stealth RNAi Collection comprises of human or mouse target genes arrayed into 96-well plates. Each plate contains ~50-80 Stealth RNAi, a p53 Positive Control Stealth RNAi, 3 negative control Stealth RNAi, the BLOCK-iT Fluorescent Oligo, and blank wells for user designed controls arranged in three configurations. The three plate configurations, Set A, Set B, and Set C, as shown in the figure below were generated by arraying the control Stealth RNAi in wells H9-12 (Set A), in wells H1-4 (Set B), or in wells H5-8 (Set C). 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 A A B B C C D D E E F F G G H H A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 Set A Set B Stealth RNAi BLOCK-iT Fluorescent Oligo Positive and negative controls Blank well (for user controls) Set C 5

Plate Layout and Control Information, Continued Plate Layout, continued The BLOCK-iT Fluorescent Oligo is arrayed in a different position for each of the plates for Set A, Set B, and Set C. In Set A, BLOCK-iT Fluorescent Oligo is arrayed in wells H1-8, in wells H5-12 for Set B, and in wells H1-4 or H9-12 for Set C (see plate layout tab in the CD-ROM for details) to allow easy identification of each plate. Plate Set A, B, and C contain Stealth RNAi duplex 1, 2, and 3 for each target gene, respectively, in the same position on each plate. For example, duplex 1, 2, and 3 for the target gene, protein kinase X, are in Plate 1A-well A10, Plate 1Bwell A10, and Plate 1C-well A10, respectively. The number on the plate represents the lot number of the plate. p53 Positive Control Each Stealth RNAi Collection Plate includes a p53 Positive Control Stealth RNAi (see plate layout on page 5) and is used in an RNAi experiment to target the human p53 gene (symbol: TP53; name: tumor protein p53; Gene ID: 7157) or the mouse p53 gene (symbol: Trp53; name: tumor protein p53; and Gene ID: 22059). The human and mouse p53 Stealth RNAi produces >70% knockdown of the target gene using a quantitative RT-PCR (qrt-pcr) assay. The p53 Positive Control Stealth RNAi is indicated as p53 in the spreadsheet on the CD. 6

Plate Layout and Control Information, Continued Negative Controls Each of the Stealth RNAi Collection Plates include three Stealth RNAi Negative Control Duplexes (see plate layout on page 5) and are ideal for use in RNAi experiments as a control for sequence independent effects following Stealth RNAi delivery in any vertebrate cell line. Each Stealth RNAi Negative Control Duplex is designed to minimize sequence homology to any known vertebrate transcript, does not induce the interferon-mediated stress response pathways as measured by real-time quantitative RT-PCR, and demonstrates minimal knockdown of vertebrate target genes. The Stealth RNAi Negative Control Duplexes differ from one another in their GC content as listed in the table below and are indicated as Lo GC Neg, Med GC Neg, and Hi GC Neg in the spreadsheet on the CD. Stealth RNAi Negative Control Duplex % GC Suitable for use with Stealth RNAi duplexes containing % GC Low GC 36 35-45% Medium GC 48 45-55% High GC 68 55-70% 7

Plate Layout and Control Information, Continued BLOCK-iT Fluorescent Oligo Each Stealth RNAi Collection Plate includes the BLOCKiT Fluorescent Oligo (see plate layout on the previous page) which allows strong, easy fluorescence-based assessment of dsrna oligomer uptake into mammalian cells. The BLOCK-iT Fluorescent Oligo is indicated as Fluorescent Ctrl in the spreadsheet on the CD. The Oligo possesses the following characteristics: Is a fluorescein-labeled, double-stranded RNA duplex with the same length, charge, and configuration as standard sirna. Contains chemical modifications that enhance the stability and allow assessment of fluorescence signal for a significantly longer time period than is obtained with other unmodified, fluorescently labeled RNA. Example: Fluorescence signal is readily detectable in HEK293 cells for at least 72 hours. Note that the strength of the fluorescence signal depends on the transfection efficiency, growth rate of the cells, and the amount of oligomer transfected. The sequence of the BLOCK-iT Fluorescent Oligo is not homologous to any known gene, ensuring against induction of non-specific cellular events caused by introduction of the Oligo into cells. Localizes primarily to the nucleus upon uptake (Fisher et al., 1993). Optimized for use with Lipofectamine 2000 transfection reagent. Important The BLOCK-iT Fluorescent Oligo is not recommended for assessing transfection efficiency using Lipofectamine RNAiMAX transfection reagent. To qualitatively assess transfection efficiency for Lipofectamine RNAiMAX transfections, we recommend using a KIF11 Stealth Select RNAi. KIF11 encodes for the motor protein kinesin family member 11, which is involved in spindle dynamics during cell mitosis. Knocking down this gene results in a roundedup phenotype after 24 hours due to a mitotic arrest (Weil et al., 2002). For ordering information, see Accessory Products, page vi. 8

CD-ROM Information Introduction Detailed gene information, gene ID (accession numbers), and the location of each Stealth RNA in each plate is included with the CD-ROM shipped with the kit. Brief introduction to the information on the CD is provided below. The RNAi sequence information is not included on the CD. To request RNAi sequence information, contact Technical Service (page 32). Information Format The information is provided as a spreadsheet in Microsoft Excel which allows you to import the information into any database or software of choice for easy data analysis and interpretation. The information in each tab of the spreadsheet is described below. Content Tab The Content Tab describes the contents of the entire Stealth RNAi Collection and includes: Gene ID Gene ID is a unique identifying number assigned to each gene in NCBI's database (GenBank, RefSeq) and is similar to LocusLink ID. Visit www.ncbi.nlm.nih.gov/locuslink for more information. VHS/HSS/MSS identifier which identifies the RNAi duplex as a Validated Human Stealth (VHS), Human Stealth Select (HSS), or Mouse Stealth Select (MSS) RNAi Gene Symbol Definition (gene name) Plate Maps- Gene ID Tab The Plate Maps-Gene ID Tab shows the location of each target gene on the plate and lists the gene ID. Note that Plate Set A, B, and C contain duplex 1, 2, and 3 for each target gene, respectively, in the same position on each plate (see page 5 for plate layout). Row H in each plate contains controls and blank wells. 9

CD-ROM Information, Continued Plate Maps- Duplex ID Tab The Plate Maps-Duplex ID Tab shows the location of each duplex on the plate and lists the VHS, MSS, or HSS identifier for each duplex. Row H shows the position of the control Stealth RNAi duplexes that are arrayed in three configurations, resulting in Plate Set A that contains controls in wells H9-12, Plate Set B that contains the controls in wells H1-4, and Plate Set C that contains the controls in wells H5-8 (see page 5 for plate layout). Gene ID- Duplex ID List Tab The Gene ID-Duplex ID List Tab lists the duplex ID (as VHS/HSS/MSS identifier), Gene ID and the location of each duplex on the plate. This list is useful for identifying the target genes (hits) and reordering the duplex from Invitrogen for further studies. 10

Experimental Overview Experimental Outline The experimental outline for using the Stealth RNAi Collection is shown below. Dilute Stealth TM RNAi Prepare transfection reagent: Lipofectamine TM 2000 or Lipofectamine TM RNAiMAX Combine diluted transfection reagent with diluted Stealth TM RNAi Prepare cells Add cells in complete medium to the transfection reagent -Stealth TM RNAi complex Perform screening assay after 24-96 hours Analyze data to identify hits Reorder Stealth TM RNAi to perform additional validation experiments 11

General Guidelines Methods Introduction General guidelines for using the Stealth RNAi Collection are described in this section. Review the information in this section prior to performing screening experiments to obtain the best results. To use the Stealth RNAi Collection effectively, we recommend that you have a working knowledge of the RNAi pathway, designing appropriate screening assays, performing high-throughput transfections and screening assays, and analyzing screening assay data to identify significant hits. Handling the Plates The Stealth RNAi Collection is supplied as a set of 96-well plates that are sealed with a removable lid. To avoid any RNase contamination and prevent any crosscontamination of wells, handle the plate with care as below: To ensure that the plate contents are not contaminated with RNase Use RNase-free sterile pipette tips and supplies for all manipulations. Wear gloves when handling plates, reagents, and solutions. Store the plate at -20ºC when not in use Aliquot the required amount of the Stealth RNAi into a sterile, RNase-free 96-well plate as described on page 20 Avoid repeated freezing and thawing of the plate Factors Affecting Gene Knockdown Levels Many factors influence the degree to which expression of a gene of interest is reduced (i.e. gene knockdown) in an RNAi experiment including: Transfection efficiency Transcription rate of the gene of interest Protein stability Growth characteristics of your mammalian cell line Take these factors into account when designing your transfection and RNAi experiments. 12

General Guidelines, Continued Cell Lines The Stealth RNAi Human Collection is designed for use with human cell lines only. Using non-human cell lines will not produce the desired level of target gene knockdown. The Stealth RNAi Mouse Collection is designed for use with mouse cell lines only. Using non-mouse cell lines will not produce the desired level of target gene knockdown. Transfection Methods You can choose any transfection method or transfection reagent suitable for delivery of dsrna oligomers to human cells for transfection. To perform transfection experiments using the Stealth RNAi Collection, we recommend using the reverse transfection protocol (page 1815) with Lipofectamine 2000 Reagent or Lipofectamine RNAiMAX Reagent. Both Lipofectamine 2000 and Lipofectamine RNAiMAX are excellent reagents to transfect Stealth RNAi. Below and on the next page the characteristics of these two reagents are summarized; use the reagent that best suits your cell line, current experiment, as well as your future plans. Lipofectamine 2000 Reagent Lipofectamine 2000 Reagent (see page v for ordering information) is a proprietary, cationic lipid-based formulation suitable for delivery of Stealth RNAi and the BLOCK-iT Fluorescent Oligo to mammalian cells for RNAi analysis (Gitlin et al., 2002; Yu et al., 2002). Using Lipofectamine 2000 to transfect eukaryotic cells offers the following advantages: Provides high transfection efficiency in a wide variety of mammalian cell types Is an outstanding reagent for transfection of sirna, plasmid DNA, as well as sirna-dna cotransfections Allows direct addition of the Stealth RNAi- Lipofectamine 2000 complexes to cells in culture medium in the presence of serum Removal of complexes or medium change or addition following transfection is not required, although complexes can be removed after 4-6 hours without loss of activity 13

General Guidelines, Continued Invitrogen offers the BLOCK-iT Transfection Optimization Kit (page v) to help you optimize RNAi transfection with Lipofectamine 2000 Reagent using controls for transfection and viability. The kit includes a Stealth RNAi molecule targeting the human p53 gene for use as a positive control and a Scrambled Stealth RNAi molecule for use as a negative control, a BLOCK-iT Fluorescent Oligo, for use as an indicator of transfection efficiency in RNAi experiments with Stealth RNAi, and Lipofectamine 2000 Reagent for highly efficient delivery of dsrna oligomers to a wide variety of mammalian cells. Lipofectamine RNAiMAX Reagent Lipofectamine RNAiMAX (see page v for ordering information) is a proprietary, cationic lipid-based formulation specifically developed for the transfection of sirna and Stealth RNAi duplexes into eukaryotic cells. Lipofectamine RNAiMAX provides the following advantages: High transfection efficiencies in many cell types to minimize background expression from untransfected cells and maximize knockdown Minimal cytotoxicity and low concentrations of RNAi duplexes reduce non-specific effects and cellular stress A broad peak of optimal transfection activity with minimal cytotoxicity, allowing achievement of high knockdown levels despite differences in cell density, minor pipetting inaccuracies, and other variations Allows direct addition of the Stealth RNAi- Lipofectamine RNAiMAX complexes to cells in culture medium in the presence of serum Removal of complexes, or medium change or addition following transfection is not required, although complexes can be removed after 4-6 hours without loss of activity 14

General Guidelines, Continued Reverse Transfection The Stealth RNAi arrayed on the Stealth RNAi Collection plate is compatible for screening using the reverse transfection protocol. The reverse transfection protocol is a fast, easy, and highthroughput method of transfection compared to traditional transfection methods and involves simultaneous addition of cells and transfection reagent to the RNAi on the plate without any prior plating of cells. See page 18 for a protocol. Optimizing Transfection Before proceeding with the screening experiment, it is important to have an optimized transfection protocol to obtain the best results. Consider the following parameters during optimization: Transfection Reagent Choose a transfection reagent that provides high transfection efficiency with low non-specific effects in your specific cell line. Transfection protocol Choose a transfection protocol that is suitable for highthroughput samples Transfection Conditions Optimize transfection conditions such as cell number, amount of the transfection reagent and RNAi, and the time period to assay for target gene knockdown. 15

General Guidelines, Continued Performing an Assay for Target Gene Knockdown To validate your Stealth RNAi, you must measure the effect of the Stealth RNAi on the target mrna using a screening assay. A screening assay usually measures the target mrna expression or target gene expression depending on the assay. Examples of screening assays include: Cell proliferation assays using the CyQUANT Cell Proliferation Assay Kit (page v) Quantitative RT-PCR (qrt-pcr) Signal transduction pathway analysis using CellSensor Cell Lines (page v) Protein expression assay using enzyme activity, immunocytochemistry, or ELISA Prior to using the Stealth RNAi Collection, select a suitable screening assay to measure gene knockdown and optimize the assay to obtain the best results. Choose a screening assay with the following criteria: High sensitivity Low background Signal stability Fast, efficient, easy to perform, and compatible with high-throughput procedures If you are measuring protein levels to analyze the Stealth RNAi-mediated inhibition, any pre-existing pool of the protein must be degraded. If the protein of interest has a long half-life, you may need to perform long-term transfection experiments (i.e. perform multiple cycles of transfection) to observe effects at the protein level. Note: The Stealth RNAi may be effective at decreasing mrna levels of the target gene; however, may not affect protein levels if the target protein has a long half-life. If possible, measure mrna and protein level to confirm the effect of Stealth RNAi on gene knockdown. 16

General Guidelines, Continued Appropriate Controls When performing Stealth RNAi analysis, it is important to include proper positive and negative controls to help evaluate your results. The p53 positive and three negative controls are included in the Stealth RNAi Collection plate for your convenience. For Lipofectamine 2000: use the BLOCK-iT Fluorescent Oligo arrayed on the plate as an indicator of transfection efficiency with Stealth RNAi using any fluorescence microscope with a standard FITC filter set. For Lipofectamine RNAiMAX: use a KIF11 Stealth Select RNAi as described on page 8. Dispense the KIF11 RNAi in one of the blank wells included on the plate. In addition, the blank wells can be used for your assay specific controls and controls for mock or untransfected cells. High- Throughput Guidelines The Stealth RNAi Collection is arrayed in 96-well plates allowing you to perform the screening using highthroughput procedures. The plates are compatible for use with a multichannel pipettor, or 8-, 12-, or 96-tip robotic loading devices with an automated liquid handling workstation. To use the plates with an automated workstation, you will need: A liquid handling robotic workstation Appropriate tips for liquid dispensing and aspiration Suitable shaker Once you have the required hardware, you will need to configure the deck of your liquid handling robot appropriately to process samples. You may use any suitable configuration of your choice. 17

Reverse Transfection Protocol Introduction Instructions for performing the reverse transfection protocol using Lipofectamine 2000 Reagent or Lipofectamine RNAiMAX Reagent are described in this section. Use this protocol as a starting point and based on your initial results, optimize the protocol. If you have an optimized reverse transfection protocol for your cell type, use the optimized protocol. Important Review the information on page 12 prior to performing the screening. You need to have an optimized transfection reagent and protocol for your cell type, and a screening assay before performing the screening. General Guidelines for Transfection Follow these general guidelines when using Lipofectamine 2000 Reagent or Lipofectamine RNAiMAX Reagent to reverse transfect Stealth RNAi into mammalian cells. Use low-passage cells, and make sure that cells are healthy and greater than 90% viable before transfection. The reverse transfection protocol is optimized for adherent cells. If you are using suspension cells, you may need to experimentally determine the optimal protocol. Do not add antibiotics to the medium during transfection as this reduces transfection efficiency and causes cell death. For optimal results, use Opti-MEM I Reduced Serum Medium to dilute transfection reagent and Stealth RNAi prior to complex formation. To increase accuracy and reduce assay variability, we recommend performing triplicate transfections for each sample condition. Always mix the Stealth RNAi stock solution thoroughly before use. Thaw and spin to collect fluid before removing sample. Assay the gene knockdown levels at a minimum of 24-72 hours. 18

Reverse Transfection Protocol, Continued Materials Needed You will need the following materials: Cell line of interest (human cell line for Stealth Human Collection and mouse cell line for Stealth Mouse Collection) Lipofectamine 2000 Reagent or Lipofectamine RNAiMAX Reagent (store at 4ºC until use, page v) Opti-MEM I Reduced Serum Medium (pre-warm to 37ºC before use, page v) Stealth RNAi Collection Plates (supplied) 96-well tissue culture plate for aliquoting the Stealth RNAi Appropriate tissue culture plates and supplies Appropriate user designed controls for blank wells Optional: For transfections with Lipofectamine RNAiMAX Reagent, a KIF11 Stealth Select RNAi. Optional: multichannel pipettors or automated liquid handling workstation Amount of Stealth RNAi to Transfect The amount of Stealth RNAi, Stealth Positive and Negative Control RNAi, BLOCK-iT Fluorescent Oligo, and your assay specific control sirna required to achieve optimal target gene knockdown must be determined experimentally for each human cell line. The recommended starting amount of each duplex used for transfection is listed in the table below: Duplex Lipofectamine 2000 transfections Stealth RNAi 100 nm 10 nm Stealth Positive 100 nm 10 nm Control RNAi (p53) Stealth Negative Control RNAi 100 nm 10 nm Lipofectamine RNAiMAX transfections BLOCK-iT Fluorescent Oligo KIF11 Stealth Select RNAi 100 nm -- -- 10 nm Based on the initial results, you may need to optimize transfection conditions as described on page 24. 19

Reverse Transfection Protocol, Continued Using the Collection Each well of the plate contains 2 nmol of Stealth RNAi sufficient to perform ~200 transfections depending on your transfection reagent. The Stealth RNAi is supplied in solution at a concentration of 20 µm. To perform your screening experiment, you need to aliquot the required amount of Stealth RNA into a sterile, RNasefree 96-well plate in a tissue culture hood to prevent any contamination. Thawing the Collection To use the collection: 1. Thaw the Stealth RNAi Collection plate at 4ºC. 2. Centrifuge the plate briefly at 4ºC to allow the contents to settle at the bottom of the well. 3. Gently remove the lid from the plate taking care to avoid any splashing of liquid into other wells that may result in sample cross contamination. Note that the lid is reusable and is required to reseal the plate. Do not discard the lid. Dispensing the Collection for Lipofectamine 2000 For Lipofectamine 2000 transfections (using a final Stealth RNAi concentration of 100 nm during transfection): 1. Transfer 0.75 µl Stealth RNAi from each well of the Stealth RNAi Collection plate to a sterile, RNase-free 96-well plate containing 25 µl Opti-MEM I Reduced Serum Medium in each well in a tissue culture hood. 2. Be sure to aliquot appropriate amount of your assay specific control RNAi to the plate containing Stealth RNAi. 3. Mix well and keep the plate on ice until use. 4. Seal the Stealth RNAi Collection plate with the lid. Make sure the lid is oriented properly (use the alphanumeric markings on the lid for proper alignment). 5. Return the Stealth RNAi Collection plate to -20ºC immediately after use. 20

Reverse Transfection Protocol, Continued Dispensing the Collection for Lipofectamine RNAiMAX For Lipofectamine RNAiMAX transfections (using a final Stealth RNAi concentration of 10 nm during transfection): 1. Transfer 0.6 µl Stealth RNAi from each well of the Stealth RNAi Collection plate to a sterile, RNase-free 96-well plate containing 100 µl Opti-MEM I Reduced Serum Medium in each well in a tissue culture hood. Mix well. 2. Be sure to aliquot 0.6 µl KIF11 Stealth Select RNAi and appropriate amount of assay specific control RNAi to the plate containing the diluted Stealth RNAi. Mix well. 3. Transfer 10 µl of the diluted Stealth RNAi and controls from each well to new sterile, RNase-free 96-well plates (we recommend at least three plates for triplicates). 4. Keep the plates on ice until use. 5. Seal the Stealth RNAi Collection plate with the lid. Make sure the lid is oriented properly (use the alphanumeric markings on the lid for proper alignment). 6. Return the Stealth RNAi Collection plate to -20ºC immediately after use. 7. Discard the plate containing the diluted Stealth RNAi after use. Preparing Cells Prepare the cells for transfection, immediately before preparing the transfection reagent or during the incubation of the transfection reagent with the Stealth RNAi (next page). 1. Harvest healthy, adherent cells by trypsin treatment. 2. Resuspend the cells in complete growth medium (with serum but without antibiotics): ~2 x 10 5 cells/ml for Lipofectamine 2000 transfections ~5 x 10 4 cells/ml for Lipofectamine RNAiMAX transfections 3. Keep the cells at 37ºC until use (do not maintain the cells in this state for more than 20-30 minutes). 21

Reverse Transfection Protocol, Continued Using Lipofectamine 2000 This reverse transfection protocol is optimized using 100 nm Stealth RNAi and Lipofectamine 2000 for a CellSensor cell line and may need some optimization based on cell line and screening assay. The recommended reagent volumes to use for transfection are listed for each well of a 96-well plate. Scale up the reagent volumes accordingly, to prepare reagents for multiple plates. The complexes are prepared inside the wells, after which cells in medium are added. Remember to include proper positive and negative controls in your experiment. 1. Add the following components to a sterile conical tube at room temperature: Lipofectamine 2000 Reagent 0.25 µl Opti-MEM I Reduced Serum Medium 25 µl 2. Mix gently and incubate at room temperature for 5 minutes. 3. Dispense 25 µl of the diluted Lipofectamine 2000 Reagent (Step 1, above) to each well of the plate containing the 25 µl of diluted Stealth RNAi (Step 4, page 20) to obtain a final volume of 50 µl. Mix gently. 4. Incubate the mixture for 15 minutes at room temperature to allow complex formation to occur. 5. Add 100 µl complete growth medium without antibiotics containing 2 x 10 4 mammalian cells (mix the cells gently to resuspend any cells that have settled before use) to each well on the plate from Step 3 to obtain a final volume of 150 µl. Mix gently by rocking the plate back and forth. The final Stealth RNAi concentration is 100 nm. 6. Incubate the plate at 37ºC in a CO2 incubator for 24-96 hours or until you are ready to assay for gene knockdown. It is not necessary to remove the complexes or change the medium; however growth medium may be replaced after 4-6 hours without any loss of transfection activity. 7. Perform the desired screening assay (page 16). Guidelines for detecting p53 and BLOCK-iT Fluorescent Oligo are on page 25. 8. Analyze data to identify hits (page 26). 22

Reverse Transfection Protocol, Continued Using Lipofectamine RNAiMAX This reverse transfection protocol is optimized using 10 nm Stealth RNAi and Lipofectamine RNAiMAX for a CellSensor cell line and may need some optimization based on cell line and screening assay. The recommended reagent volumes to use for transfection are listed for each well of a 96-well plate. Scale up the reagent volumes accordingly, to prepare reagents for multiple plates. The complexes are prepared inside the wells, after which cells in medium are added. Remember to include proper positive and negative controls in your experiment. 1. Add the following components to a sterile conical tube at room temperature: Lipofectamine RNAiMAX Reagent 0.2 µl Opti-MEM I Reduced Serum Medium 10 µl 2. Mix gently. 3. Dispense 10 µl of the diluted Lipofectamine RNAiMAX Reagent (Step 1, above) to each well of the plate containing the 10 µl of diluted Stealth RNAi (Step 4, page 21) to obtain a final volume of 20 µl. Mix gently. 4. Incubate the mixture for 10-20 minutes at room temperature to allow complex formation to occur. 5. Add 100 µl complete growth medium without antibiotics containing 5 x 10 3 mammalian cells (mix the cells gently to resuspend any cells that have settled before use) to each well on the plate from Step 3 to obtain a final volume of 120 µl. Mix gently by rocking the plate back and forth. The final Stealth RNAi concentration is 10 nm. 6. Incubate the plate at 37ºC in a CO2 incubator for 24-96 hours or until you are ready to assay for gene knockdown. It is not necessary to remove the complexes or change the medium; however growth medium may be replaced after 4-6 hours without any loss of transfection activity. 7. Perform the desired screening assay (page 16). Guidelines for detecting p53 and KIF11 knockdown are on page 25. 8. Analyze data to identify hits (page 26). 23

Reverse Transfection Protocol, Continued Optimizing Lipofectamine 2000 Transfections 0.25 µl Lipofectamine 2000 Reagent per well is recommended as a starting point but optimization of transfection conditions may be required. A range of 0.125 µl to 1 µl Lipofectamine 2000 Reagent is recommended. 100 nm Stealth RNAi is recommended as a starting point but you can use a range of 10-150 nm Stealth RNAi for optimization. Note that for RNAi molecules inducing >90% target knockdown, the amount of dsrna required to obtain effective knockdown may be less than the amount specified. This needs to be determined empirically for each cell line. Optimizing Lipofectamine RNAiMAX Transfections 0.2 µl Lipofectamine RNAiMAX Reagent per well is recommended as a starting point but optimization of transfection conditions may be required. A range of 0.05 µl to 0.3 µl Lipofectamine RNAiMAX Reagent is recommended. 10 nm Stealth RNAi is recommended as a starting point but you can use a range of 1-50 nm Stealth RNAi for optimization. Note that for RNAi molecules inducing >90% target knockdown, the amount of dsrna required to obtain effective knockdown may be less than the amount specified. This needs to be determined empirically for each cell line. For extended time course experiments (> 72 hours), consider a cell density that is 10-20% confluent 24 hours after plating (~2 x 10 3 cells per well). 24

Detecting Control Signal Introduction p53 mrna or Protein Fluorescence Signal This section provides instructions for detecting the signals of the p53 Positive Control Stealth, RNAi BLOCK-iT Fluorescent Oligo and KIF11 Stealth Select RNAi controls You may use any method of choice to detect human p53 expression levels after treatment with the positive control Stealth RNAi. To assay for p53 mrna levels, we recommend performing qrt-pcr using Invitrogen s custom LUX primers. Use the LUX Designer available at www.invitrogen.com/lux to help you design and order suitable primers to use for qrt-pcr analysis. To prepare mrna from treated or untreated cells, use Invitrogen s mrna Catcher PLUS Kit (page v). When performing qrt-pcr, remember to normalize results to an internal control RNA (e.g. β-actin, GAPDH, or cyclophilin). To assay for p53 protein levels, we recommend performing Western blot analysis using a suitable antibody to human p53. Remember to take into account the half-life of the protein when assessing RNAi effects at the protein level. Once you have transfected your mammalian cells with the BLOCK-iT Fluorescent Oligo, you may qualitatively assess Oligo uptake using any fluorescence microscope and any standard FITC filter set (λex = 494 nm, λem = 519 nm) to detect the fluorescence signal from the BLOCK-iT Fluorescent Oligo. Uptake of the fluorescent oligomer by at least 80% of cells correlates with high levels of gene knockdown by effective Stealth RNAi. KIF11 Knockdown Phenotype KIF11 encodes for a motor protein of the kinesin like family involved in chromosome positioning, centrosome separation and establishing a bipolar spindle during cell mitosis. Adherent cells in which KIF11/Eg5 is knocked down exhibit a rounded-up phenotype after 24 hours due to a mitotic arrest (Weil et al., 2002); slow growing cells may take up to 72 hours to display the rounded phenotype. Alternatively, growth inhibition can be assayed after 48-72 hours. 25

Data Analysis Guidelines Introduction General guidelines for analyzing the screening data to identify significant hits are described in this section. You can use a spreadsheet program with standard statistical tools such as Microsoft Excel or commercially available software designed with complex statistical algorithms for data analysis and identifying significant hits. Performing data analysis requires a certain degree of expertise with statistics and Excel or another spreadsheet program. Data Analysis Depending on the type of assay that you performed, the data may be from a colorimetric, fluorescent, or luminescent read out. Analyze the data to identify significant hits. The number of hits obtained will depend on the target genes and the type of screening assay used. Data analysis guidelines using Excel are described below. 1. Subtract the background or values from blank wells from all treatments. 2. Calculate the average and standard deviation for the negative controls to determine the baseline phenotype. 3. Calculate the average for each set of duplicate or triplicate transfection (duplicate or triplicate transfection is recommended per duplex) and normalize to the average of the negative controls. 4. Set the hit threshold at 2-3 standard deviations from the average of the negative controls and define the hits. Alternatively, calculate the average and standard deviation for all the RNAi treatments except the negative controls. 5. Calculate the average for each set of duplicate or triplicate transfection and normalize to the average of all the RNAi treatments except the negative controls. 6. Set the hit threshold at 2-3 standard deviations from average of all the RNAi treatments except the negative controls and define the hits. Values 2-3 standard deviations above or below the average of the negative controls or all RNAi treatments, depending on the method chosen are considered significant hits. 26

Data Analysis Guidelines, Continued The Next Step Once you have identified a potential hit, we recommend performing secondary screening using identical conditions to confirm the observed phenotype. Confirm the reduction in target mrna or protein level using additional methods. Stealth RNAi is available in 20 nmole scale from Invitrogen to perform additional experiments that allow further validation (see next page). 27

Reordering Stealth RNAi Introduction Each Stealth RNAi is available for reorder at a scale of 20 nmole or higher. See instructions below to reorder the duplex. Stealth RNAi Collection users can take advantage of special pricing by using a promotion code during the reordering process. Contact Technical service (page 32) to obtain the promotion code. Information Needed You will need the following information to reorder the Stealth RNAi: HSS, MSS, or VHS identifier number for your specific Stealth RNAi (refer to the Gene ID-Duplex ID List Tab on the spreadsheet included on the CD) Special Pricing Quote Number from Technical Service (page 32) to obtain special pricing, if you are a Stealth RNAi Collection user 28

Reordering Stealth RNAi, Continued Stealth Quick Order Site 1. To reorder Stealth RNAi, go to www.invitrogen.com/stealthquickorder 2. The Stealth Quick Order window opens. 3. Enter the HSS (Human Stealth Select), MSS (Mouse Stealth Select), or VHS (Validated Human Stealth ) number in the field. To obtain this number for your specific Stealth RNAi, refer to the spreadsheet included on the CD (Gene ID-Duplex ID List Tab). 4. Select Go to Order form to open the order form. 5. Enter the ordering information and special pricing code (obtained from Technical Service) in the Quote Number field. 6. Select Order to order the Stealth RNAi. 29

Troubleshooting Introduction Review the information below to troubleshoot your experiments with the Stealth RNAi Collection. To troubleshoot your screening assay, refer to the manual supplied with the assay. Problem Cause Solution Low gene knockdown Different level of gene knockdown obtained with the three RNAi for the same target gene Poor transfection efficiency Transfection reagent may be toxic Cells not healthy or non-species specific cell line used Transfection Reagent handled incorrectly The three duplexes may have differences in the potency Optimize the transfection conditions as described on page 15. Be sure to assess the transfection efficiency using BLOCK-iT Fluorescent Oligo or KIF11. Use the standard transfection protocol instead of reverse transfection protocol. Use the minimum amount of transfection reagent required to produce the optimal signal. We recommend using Lipofectamine 2000 or Lipofectamine RNAiMAX Reagent for transfection. Do not allow the cells to remain in medium containing the transfection reagent. Replace the medium with fresh medium after 4-10 hours. Use another transfection reagent that does not product any toxic effects. Be sure to use healthy, >90% viable human or mouse cells for transfection. Use cells that have undergone fewer passages. Store at +4 C. Do not freeze. Mix gently by inversion before use. Do not vortex. Perform a secondary screening and additional validation experiments to confirm the observed phenotype and knockdown. 30

Troubleshooting, Continued Problem Cause Solution High rate of false positives Identified very few hits High variability Screening assay not designed properly Poor transfection efficiency Stringent limits set for the screening assay Inconsistent assay conditions Cells not healthy Used incorrect cell number for each well Select a screening assay that is very specific rather than using broad screening assays such as cell proliferation. Adjust the limits of the screening assay to produce significant hits. Perform a secondary screening with different RNAi duplexes to reduce the rate of false positives. Optimize the transfection conditions as described on page 15. Be sure to measure the transfection efficiency using BLOCK-iT Fluorescent Oligo or KIF11 Stealth Select RNAi. Use the standard transfection protocol instead of reverse transfection protocol. Lower the limits of the screening assay to produce significant hits. Be sure the controls are set correctly to result in correct data analysis. Be sure to use the same conditions for assay as well as transfection parameters between each screening experiment to ensure reproducibility. Be sure to use healthy, >90% viable cells for transfection. Use cells that have undergone fewer passages. Mix the cells gently to resuspend any cells that have settled or adhered to ensure that same amount of cells are transferred to each well of the plate. Check to make sure there are no errors during cell counting. 31

Technical Service Appendix Web Resources Visit the Invitrogen Web site at www.invitrogen.com for: Technical resources, including manuals, vector maps and sequences, application notes, MSDSs, FAQs, formulations, citations, handbooks, etc. Complete technical service contact information Access to the Invitrogen Online Catalog Additional product information and special offers Contact Us For more information or technical assistance, call, write, fax, or email. Additional international offices are listed on our Web page (www.invitrogen.com). Corporate Headquarters: European Headquarters: Invitrogen Corporation Invitrogen Ltd 1600 Faraday Avenue Inchinnan Business Park Carlsbad, CA 92008 USA 3 Fountain Drive Tel: 1 760 603 7200 Paisley PA4 9RF, UK Tel (Toll Free): 1 800 955 6288 Tel: +44 (0) 141 814 6100 Fax: 1 760 602 6500 Tech Fax: +44 (0) 141 814 6117 E-mail: tech_support@invitrogen.com E-mail: eurotech@invitrogen.com Material Safety Data Sheets (MSDSs) MSDSs (Material Safety Data Sheets) are available on our website at www.invitrogen.com/msds. 32