Transfecting Cells
Contents
1. 2ml 100 pmol in 250 pl 5 pl in 250 pl 60 mm 20 cm 5 ml 200 pmol in 0 5 ml 10 pl in 0 5 ml 10 cm 60 cm 15 ml 600 pmol in 1 5 ml 30 pl in 1 5 ml Surface areas may vary depending on the manufacturer 18 Continued on next page Transfecting Cells Continued Detecting Fluorescence The Next Step TM If you have transfected your mammalian cells with the BLOCK T Fluorescent Oligo you may qualitatively assess Oligo uptake in live cells 6 24 hours post transfection using fluorescence microscopy You may use any type of fluorescence microscope and a standard FITC filter set Xex 494 nm Aem 519 nm green for detection An example of expected results is shown on page 20 Once you have assessed the gene knockdown we recommend that you validate the results using additional methods such as qPCR Western analysis immunohistochemistry or any other functional assay TM A variety of BioModule Units that include qualified reagents and validated protocols are available from Invitrogen to perform further validation experiments page 26 19 Expected Results Example of Results An example of results obtained after performing an RNAi experiment using 20 a tubulin 50 5 kDa the BioModule Transfection and Control Unit with BLOCK iT Technology and validation using western immunodetection with the BioModule Western Analysis are shown below In this experiment A549 cells were transfe2. Do not add antibiotics to the media during transfection Plate cells such that they will be 30 50 confluent at the time of transfection e Increase the amount of RNAi duplex transfected page 18 e Optimize the transfection conditions for your cell line by varying the amount of transfection reagent used Be sure to assess the transfection efficiency using BLOCK iT Fluorescent Oligo Low levels of gene knockdown observed other causes Did not wait long enough after transfection before assaying for gene knockdown Repeat the transfection and wait for a longer period of time after transfection before assaying for gene knockdown Perform a time course of expression to determine the point at which the highest degree of gene knockdown occurs Cells not healthy or used non species specific cell lines Be sure to use healthy gt 90 viable cells Use species specific cell lines to obtain the best results Lipofectamine 2000 Reagent handled incorrectly Store the reagent at 4 C Do not freeze Mix gently by inversion before use Do not vortex Target protein is stable i e has a long half life Perform qRT PCR analysis using primers to assay for target gene knockdown at the mRNA level 22 Continued on next page Troubleshooting Continued Problem Reason Solution No gene knockdown Used a cell line that does not Be sure to use a cell line that expres
3. one of the several BioModule Units available from Invitrogen page 26 for gene expression profiling Each of the BioModule Units for gene expression profiling includes high quality reagents and validated protocols with relevant controls for each step of the workflow see below Each unit is designed to provide an integrated workflow that allows you to perform various steps seamlessly during expression analysis Gene expression profiling comprises multiple steps employing various technologies such as microarray analysis or quantitative PCR qPCR for analysis at the nucleic acid level western immunodetection and immunohistochemistry for analysis at the protein level and RNAi for functional analysis expression profiles Microarray Analysis lt _ y Quantitative Analysis of Synthetic and qPCR measurement gene Vector based RNAi Analysis of RNA function A transcripts Western Detection or IHC Staining Protein detection from cells and tissues Continued on next page Overview Continued System Components System Overview TM The BioModule Transfection and Control Unit includes TM Lipofectamine 2000 Reagent for highly efficient delivery of dsRNA oligomers into a wide variety of mammalian cells BLOCK iT Fluorescent Oligo a fluorescein labeled double stranded RNA dsRNA oligomer for use as an indicator of
4. Bird C R Ray J Schuch W and Grierson D 1990 Expression of a Truncated Tomato Polygalacturonase Gene Inhibits Expression of the Endogenous Gene in Transgenic Plants Mol Gen Genet 224 477 481 van der Krol A R Mur L A Beld M Mol J N and Stuitje A R 1990 Flavonoid Genes in Petunia Addition of a Limited Number of Gene Copies May Lead to a Suppression of Gene Expression Plant Cell 2 291 299 Voinnet O Pinto Y M and Baulcombe D C 1999 Suppression of Gene Silencing A General Strategy Used by Diverse DNA and RNA Viruses of Plants Proc Natl Acad Sci USA 96 14147 14152 Yu J Y DeRuiter S L and Turner D L 2002 RNA Interference by Expression of Short interfering RNAs and Hairpin RNAs in Mammalian Cells Proc Natl Acad Sci USA 99 6047 6052 Zamore P D 2001 RNA Interference Listening to the Sound of Silence Nat Struct Biol 8 746 750 2005 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 31
5. Oligo in 50 ul Opti MEM I Reduced Serum Medium without serum resulting concentration of the Fluorescent Oligo is 100 nM Mix gently Mix Lipofectamine 2000 gently before use then dilute 1 ul in 50 ul Opti MEM I Reduced Serum Medium Mix gently and incubate for 5 minutes at room temperature After the 5 minute incubation combine the diluted oligomer with the diluted Lipofectamine 2000 Mix gently and incubate for 20 minutes at room temperature solution may appear cloudy TM Add the oligomer Lipofectamine 2000 complexes to each well containing cells and medium Mix gently by rocking the plate back and forth Incubate the cells at 37 C in a CO incubator for 24 96 hours until you are ready to assay for gene knockdown Medium may be changed after 4 6 hours Optimizing To obtain the highest transfection efficiency and low non specific effects optimize Transfection transfection conditions as described below TM e Usea range of 0 5 ul to 1 5 ul Lipofectamine 2000 Reagent for 24 well format e Use a range of 20 100 nM Stealth RNAi or siRNA for 24 well format e Depending on the nature of the target gene you may transfect cells at higher densities Kit Contents and Storage Shipping and The shipping conditions for each component are listed in the table below Upon Storage receipt store the components as described below Components Shipping Storage Lipofectamine
6. Stealth RNAi or siRNA 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 or siRNA on gene knockdown Continued on next page General Guidelines Continued Appropriate Controls When performing RNAi analysis it is important to include proper positive and negative controls to help evaluate your results The BLOCK iT Fluorescent Oligo and Stealth RNAi Negative Control Duplex are included in the BioModule Transfection and Control Unit for your convenience Use the BLOCK iT Fluorescent Oligo as an indicator of transfection efficiency with Stealth RNAi or siRNA using any fluorescence microscope with a standard FITC filter set Be sure to include your assay specific controls and controls for mock or untransfected cells 13 Transfecting Cells Introduction SECOS se Z 25 S MI Handling the Stealth RNAi Negative Control Duplex 14 This section provides guidelines to transfect the Stealth RNAi or siRNA into the mammalian cell line of interest to perform RNAi analysis e We recommend Opti MEM I Reduced Serum Medium to dilute Lipofectamine 2000 Reagent and RNAi duplexes before complexing e Do not add antibiotics to media during transfection as this causes cell death e Use low passage cells a
7. as appropriate should be determined experimentally for each mammalian cell line The recommended starting amount of each duplex used for transfection is listed in the table below Duplex Recommended Conc Stealth RNAi or siRNA 40 nM Stealth RNAi Negative Control Duplex 40 nM BLOCK iT Fluorescent Oligo 100 nM Based on the initial results you may need to optimize transfection conditions as described on page 18 You will need the following materials e Mammalian cell line of interest cultured in the appropriate growth medium e Lipofectamine 2000 Reagent supplied with the kit store at 4 C until use e Opti MEM I Reduced Serum Medium supplied with the kit pre warm to 37 C before use e Stealth RNAi or siRNA duplex 20 uM in 1X RNA Annealing Buffer e BLOCK iT Fluorescent Oligo supplied with the kit 20 uM in 1X RNA Annealing Buffer e Stealth RNAi Negative Control Duplex supplied with the kit 20 uM in 1X RNA Annealing Buffer e Appropriate tissue culture plates and supplies e Appropriate user designed controls Continued on next page Transfecting Cells Continued Note Transfection Protocol Use the transfection procedure described below as a starting point optimize transfections as described in Optimizing Transfection page 18 especially if you are transfecting a mammalian cell line for the first time To reduce well to well variability when transfecting multiple repli
8. before assaying for fluorescence Low transfection efficiency See previous page for details 23 Appendix Technical Service Web Resources Visit the Invitrogen Web site at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical service contact information e Access to the Invitrogen Online Catalog e 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 Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 1600 Faraday Avenue LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail E mail E mail tech_service invitrogen com jpinfo invitrogen com eurotech invitrogen com Material Data Safety Sheets MSDSs Limited Warranty 24 MSDSs are available on our Web site at www invitrogen com On the home page click on Technical Resources and follow instructions on
9. 2000 Reagent since some serum free formulations e g CD293 SFM II VP SFM may inhibit cationic lipid mediated transfection TM e Thaw Stealth RNAi Negative Control Duplex stock solution on ice or at room temperature After use return to 20 C storage e Multiple freeze thaw cycles are permitted without loss of activity if stock solution is handled properly e Ensure that the stock solution does not become contaminated with RNase Continued on next page Transfecting Cells Continued Handling the BLOCK iT Fluorescent Oligo Using the BLOCK iT Fluorescent Oligo The BLOCK iT Fluorescent Oligo is supplied as a 20 uM stock solution in an annealing buffer Follow the guidelines below when handling the BLOCK iT Fluorescent Oligo stock solution e The BLOCK iT Fluorescent Oligo is light sensitive Store the stock solution at 20 C protected from light The stock solution is stable for at least 6 months if stored properly e When using thaw the stock solution on ice or at room temperature Once thawed place the tube on ice until use After use return stock solution to 20 C storage e The stock solution may be frozen and thawed multiple times without loss of fluorescence signal if handled properly e Take precautions to ensure that the stock solution does not become contaminated with RNase a Use RNase free sterile pipette tips and supplies for all manipulations b Wear gloves when handl
10. 2000 Reagent Blue ice 4 C do not freeze BLOCK iT Fluorescent Oligo Blue ice 20 C protect from light Stealth RNAi Negative Control Dry ice 20 C Duplex Medium GC Opti MEM I Reduced Serum Room 2 C to 8 C in the dark Medium temperature Kit Contents The components included with the BioModule Transfection and Control Unit with BLOCK iT Technology are described below Box Item Composition Amount 1 Lipofectamine 2000 Reagent Proprietary formulation 0 75 ml 2 3 BLOCK iT Fluorescent Oligo 20 pM stock of fluorescein labeled double 2x 125 pl stranded RNA dsRNA oligomer in 100 mM potassium acetate 30 mM HEPES KOH pH 7 4 2 mM Magnesium acetate 4 Stealth RNAi Negative Control 20 uM in 1X RNA Annealing Dilution Buffer 250 pl Duplex Medium GC 1X RNA Annealing Dilution 10 mM Tris HCl pH 8 0 20 mM NaCl 1mM 1 ml Buffer EDTA pH 8 0 5 Opti MEM I Reduced Serum See below for formulation 100 ml Medium Formulation of Opti MEM I Reduced Serum Medium is a modification of Eagle s Minimal Opti MEM Essential Medium buffered with HEPES and sodium bicarbonate and Reduced Serum supplemented with hypoxanthine thymidine sodium pyruvate L glutamine or Medium GLUTAMAX trace elements and growth factors The protein level is minimal 15 pg ml insulin and transferrin are the only protein supplements Phenol red is included at a reduced concentration as a pH i
11. 9 13084 Bernstein E Caudy A A Hammond S M and Hannon G J 2001 Role for a Bidentate Ribonuclease in the Initiation Step of RNA Interference Nature 409 363 366 Bosher J M and Labouesse M 2000 RNA Interference Genetic Wand and Genetic Watchdog Nature Cell Biol 2 E31 E36 Carrington J C and Ambros V 2003 Role of MicroRNAs in Plant and Animal Development Science 301 336 338 Cogoni C and Macino G 1997 Isolation of Quelling Defective qde Mutants Impaired in Posttranscriptional Transgene Induced Gene Silencing in Neurospora crassa Proc Natl Acad Sci USA 94 10233 10238 Cogoni C and Macino G 1999 Gene Silencing in Neurospora crassa Requires a Protein Homologous to RNA Dependent RNA Polymerase Nature 399 166 169 Cogoni C Romano N and Macino G 1994 Suppression of Gene Expression by Homologous Transgenes Antonie Van Leeuwenhoek 65 205 209 Dykxhoorn D M Novina C D and Sharp P A 2003 Killing the Messenger Short RNAs that Silence Gene Expression Nat Rev Mol Cell Biol 4 457 467 Fisher T L Terhorst T Cao X and Wagner R W 1993 Intracellular Disposition and Metabolism of Fluorescently Labeled Unmodified and Modified Oligonucleotides Microinjected into Mammalian Cells Nuc Acids Res 21 3857 3865 Gitlin L Karelsky S and Andino R 2002 Short Interfering RNA Confers Intracellular Antiviral Immunity in Human Cells Nature 418 430 434 Hammo
12. Ai duplex of choice by gene name GenBank accession number or keyword The search results are linked directly to the ordering page allowing you to easily order the RNAi molecule of choice Validated Stealth RNAi The Validated Stealth RNAi DuoPak includes two highly effective functionally tested Stealth RNAi molecules designed for in vitro and in vivo RNAi analysis of a particular human target gene Each Validated Stealth RNAi duplex is supplied in a ready to use format and targets a different region of the human gene of interest demonstrating 80 target gene knockdown Stealth Select RNAi The Stealth Select RNAi are highly effective pre designed Stealth RNAi molecules available individually or as a set of three non overlapping sequences to thousands of human mouse or rat genes For each set of three Stealth Select RNAi 2 out of 3 RNAi sequences are guaranteed to knockdown transcript levels by at least 70 Stealth RNAi Collections TM The Stealth RNAi Collections combine the power of chemical modification with advanced in silico design algorithms to provide you with an advanced RNAi collection suited for RNAi studies A large variety of Stealth RNAi Collections are available from Invitrogen TM Each Stealth RNAi 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 silen
13. SC 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 amp 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 amp Labouesse 2000 Dykxhoorn et al 2003 Hannon 2002 Plasterk amp Ketting 2000 Zamore 2001 This manual provides the following information e An overview of Stealth RNAi and the RNAi pathway e General guidelines for transfection and performing expression screening assays e Transfection protocol for transfecting the Stealth RNAi or siRNA duplex into mammalian cells for RNAi analysis e Example of expected results e Troubleshooting Protocols for screening or expression assays are not included in this manual Description of Components Introduction Lipofectamine 2000 Reagent Stealth RNAi Negative Control Duplex BLOCK iT Fluorescent Oligo TM Brief description of the components included with the BioModule Transfection and Control Unit with BLOCK iT Technology is described in this section TM Lipofectamine 2000 Reagent is a proprietary formulation for the delivery of the BLOCK iT Fluoresce
14. agent is described below Refer to this manual for a detailed protocol Preparing Grow your mammalian cell line of choice in the recommended medium Use low passage Cells cells and make sure that cells are healthy and greater than 90 viable before transfection Amount of The recommended starting amount of each duplex used for transfection is listed below RNAi to To achieve optimal target gene knockdown you may need to experimentally determine Transfect the amount of RNAi required for each mammalian cell line Duplex Recommended Conc Stealth RNAi or siRNA 40 nM Stealth RNAi Negative Control Duplex 40 nM BLOCK iT Fluorescent Oligo 100 nM TM Perform Use this procedure to transfect Stealth RNAi or siRNA into mammalian cells in a Transfection 24 well format All amounts and volumes are given on a per well basis For other formats see Scaling Up or Down Transfections page 18 Use this procedure as a starting point and optimize transfections as described below 1 One day before transfection plate cells in 500 ul of growth medium without antibiotics such that they will be 30 50 confluent at the time of transfection For each transfection sample prepare as follows a Dilute 20 pmol 1 ul of 20 uM solution Stealth RNAi or siRNA oligomer in 50 ul Opti MEM I Reduced Serum Medium without serum resulting concentration of RNA is 40 nM Mix gently Dilute 60 pmol 3 ul of 20 uM solution BLOCK iT Fluorescent
15. c cellular events caused by introduction of the Oligo into cells Localizes primarily to the nucleus upon uptake Fisher et al 1993 For examples of fluorescent uptake in adherent panel A and suspension panel B cells see the figure below Important The BLOCK iT Fluorescent Oligo is designed strictly for use as a tool for siRNA uptake assessment and is not meant to provide any information about the behavior of your Stealth RNAi or siRNA including its cellular localization half life or stability Continued on next page Description of Components Continued Opti MEM Reduced Serum Medium 1X RNA Annealing Dilution Buffer Opti MEM I Reduced Serum Medium is a versatile chemically defined medium used for diluting the lipid and nucleic acid during transfection The Opti MEM I Reduced Serum Medium is a multi purpose medium proven to be useful in reducing serum requirements for a wide variety of cell lines and applications and has been effective in the growth and maintenance of adherent and non adherent cell lines When supplemented with 2 4 fetal bovine serum or alternative sera Opti MEM I Reduced Serum Medium supports proliferative rates and maximal cell densities comparable to and in some cases superior to conventional media supplemented with 10 fetal bovine serum Relatively non fastidious cell lines may be maintained in long term culture with even more substantial serum reduction If using adh
16. cates e g triplicates proportionally scale up the reagent volumes to form complexes Step 2 below then aliquot an equal volume of complexes into each well Prepare Stealth RNAi or siRNA duplex at a concentration of 20 uM in 1X RNA Annealing Buffer 20 uM Stealth RNAi or siRNA 20 pmol l TM Use this procedure to transfect Stealth RNAi or siRNA into mammalian cells in a 24 well format All amounts and volumes are given on a per well basis For other formats see Scaling Up or Down Transfections page 18 1 One day before transfection plate cells in 500 ul of growth medium without antibiotics such that they will be 30 50 confluent at the time of transfection Note Transfecting cells at a lower density allows a longer interval between transfection and assay time and minimizes the loss of cell viability due to cell overgrowth TM For each transfection sample prepare oligomer Lipofectamine 2000 complexes as follows a Dilute 20 pmol Stealth RNAi or siRNA oligomer i e 1 ul of 20 uM Stealth RNAi or siRNA oligomer in 50 ul Opti MEM I Reduced Serum Medium without serum resulting concentration of RNA is 40 nM Mix gently b Dilute 60 pmol BLOCK iT Fluorescent Oligo i e 3 ul of 20 uM Fluorescent Oligo in 50 ul Opti MEM I Reduced Serum Medium without serum resulting concentration of the Fluorescent Oligo is 100 nM Mix gently TM c Mix Lipofectamine 2000 gently before use then dil
17. cing screening experiments Stealth RNAi is designed for use with species specific cell lines only Using non species specific cell lines will not produce the desired level of target gene knockdown For example use the Stealth RNAi designed for human targets with human cell lines only Using mouse or rat cell lines will not produce the desired level of target gene knockdown The BioModule Transfection and Control Unit includes Lipofectamine 2000 Reagent for delivery of dsRNA oligomers to mammalian cells for transfection An optimized transfection protocol for mammalian cells is described on page 17 However depending on your cell type you may need to optimize the transfection conditions such as cell number amount of the transfection reagent and RNAi and the time period to assay for target gene knockdown to obtain the best results Cell type specific RNAi transfection protocols are also available at www invitrogen com rnai Continued on next page 11 General Guidelines Continued Factors Affecting Gene Knockdown Levels Performing an Assay for Target Gene Knockdown 12 A number of factors can influence the degree to which expression of your gene of interest is reduced i e gene knockdown in an RNAi experiment including e Transfection efficiency e Transcription rate of the target gene of interest e Stability of the target protein e Growth characteristics of your mammalian cell line Take
18. cted with MAPK1 Validated Stealth RNAi Duopak 2 different duplexes targeting MAP Kinase 1 MAPK1 available from Invitrogen cat no 12935 025 or with Stealth RNAi Negative Control medium GC using Lipofectamine 2000 Reagent as described in this manual Control transfection experiments were performed with BLOCK iT Fluorescent Oligo see next page for results 48 hours post transfection cells were harvested and lysed with Cell Extraction Buffer containing the Protease Inhibitor Cocktail The lysate 8 ug was analyzed on a NuPAGE Novex Bis Tris Gel and proteins were transferred onto an Invitrolon PVDF membrane The blots were analyzed using the WesternBreeze Chemiluminescent Kit using a polyclonal rabbit anti MAPK1 ERK2 antibody cat no 71 1800 at 1 1500 dilution or a monoclonal mouse anti a tubulin antibody cat no 32 2500 at a 1 2000 dilution The results indicate effective knockdown of MAPK1 by each Validated Stealth RNAi targeting MAPK1 Anti MAPK1 ERK2 antibody Lane 1 Validated Stealth RNAi duplex 1 1 2 3 4 5 kDa Lane 2 Validated Stealth RNAi duplex 2 60 Lane 3 Stealth RNAi Negative Control 50 medium GC MAPK1 40 Lane 4 Control non transfected A549 42 kDa cells 30 Lane 5 5 pl MagicMark XP Western Protein Standard the difference in band Anti a tubulin antibody intensity for the standard is due to 1 2 3 4 5 kDa differences in exposure times Continued on next page Expec
19. direct or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose Product Qualification Introduction Lipofectamine 2000 Reagent Stealth RNAi Negative Control Duplex BLOCK iT Fluorescent Oligo Opti MEM Reduced Serum Medium Invitrogen qualifies the BioModule Transfection and Control Unit with TM BLOCK iT Technology components as described below TM Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates Sabaraud dextrose agar plates and fluid thioglycolate medium and functionally by transfection of CHO K1 cells with a reporter plasmid The Stealth RNAi Negative Control Duplex is qualified as follows e The identity and concentration of each corresponding single stranded RNA oligo is verified by mass spectrometry and optical density reading respectively e After annealing the Stealth RNAi Negative Control Duplex is analyzed by gel electrophoresis to verify its integrity and to confirm the absence of RNA degradation The BLOCK iT Fluorescent Oligo is qualified as follows e The identity and concentration of each corresponding single stranded RNA oligo is verified by mass spectrometry and optical density reading respectively e After annealing the BLOCK iT Fluor
20. e Transfection and Control Unit you need to synthesize or purchase Stealth RNAi or siRNA molecules to your target gene of interest for performing RNAi analysis To obtain highly specific effective gene knockdown and eliminate non specific effects we recommend that you use Stealth RNAi For more information on Stealth RNAi see page 3 To obtain RNAi molecules you may e Purchase pre designed Stealth RNAi see next page TM Choose from a large selection of pre designed Stealth RNAi from Invitrogen e Synthesize Stealth RNAi or siRNA duplexes TM If a Stealth RNAi molecule of choice is not available from our collection of pre designed Stealth RNAi use the BLOCK iT RNAi Designer an online tool from Invitrogen www invitrogen com rnaidesigner to help you design and order custom Stealth RNAi or traditional unmodified siRNA molecules for any target gene of interest The RNAi Designer incorporates published rules on RNAi design into a proprietary algorithm to design most effective RNAi sequences to obtain high level gene knockdown Continued on next page General Guidelines Continued Stealth RNAi Products Cell Lines Transfection TM A large variety of ready to order pre designed Stealth RNAi products are available from Invitrogen to facilitate RNAi analysis Use the BLOCK iT RNAi Express Search Engine www invitrogen com RNAiExpress to search for a Stealth RN
21. er and RNAi Express to design and order RNAi duplexes to mammalian targets OR qPCR Analysis Immuno histochemistry Analysis Western Analysis Any other functional assay Continued on next page Experimental Overview Continued Materials Needed You will need the following materials e Stealth RNAi or siRNA duplex of choice supplied by the user see page 10 for details e Lipofectamine 2000 Reagent supplied with the kit e Opti MEM I Reduced Serum Medium supplied with the kit e BLOCK iT Fluorescent Oligo supplied with the kit e Stealth RNAi Negative Control Duplex medium GC supplied with the kit e Mammalian cell line of choice supplied by the user e Cell culture media and cell culture plates supplied by the user Methods General Guidelines Introduction Note RNAi Molecules 10 General guidelines for using the BioModule Transfection and Control Unit are described in this section Review the information in this section prior to performing the transfection and screening experiments to obtain the best results TM To use the BioModule Transfection and Control Unit effectively we recommend that you have a working knowledge of the RNAi pathway designing appropriate screening assays performing transfections and screening assays and analyzing screening assay data to identify significant hits TM To use the BioModul
22. erent cell lines and less than 2 serum supplementation or in an agitated system such as in roller bottles the medium should be further supplemented with 100 mg l CaCL The versatility of Opti MEM I Reduced Serum Medium in the propagation of various cell types makes this medium the optimal choice for many cell culture requirements For details on using the medium to reduce serum requirements for various cell lines download the Opti MEM I Reduced Serum Medium manual from www invitrogen com 1X RNA Annealing Dilution Buffer is supplied with the kit for use in diluting RNAi molecules if necessary For an RNAi molecule the concentration of reagent required to induce effective target gene knockdown if the RNAi molecule is active can vary with each mammalian cell line and requires optimization When optimizing your transfection conditions use the 1X RNA Annealing Dilution Buffer to dilute the RNAi reagent stock solution if needed Experimental Overview Workflow The experimental workflow for using the BioModule Transfection and Control Unit BLOCK iT Technology is shown below OR Obtain Stealth RNAi or siRNA duplex lt Y Prepare Cells y Perform transfection using Lipofectamine 2000 Reagent Perform screening assay after 24 96 hours Analyze data Validate results using Use BLOCK T RNAi Design
23. escent Oligo is analyzed by gel electrophoresis to verify its integrity and to confirm the absence of RNA degradation Opti MEM I Reduced Serum Medium is subjected to pH osmolality endotoxin bacterial fungal and mycoplasma testing The endotoxin level must be less than 1 0 EU ml Each lot of Opti MEM I is evaluated utilizing sensitive quantitative assays for its ability to support cloning efficiency of a murine myeloma cell line and growth over multiple subcultures of an adherent cell line Test lots of Opti MEM I Reduced Serum Medium at 2 CHO growth and 4 Sp2 cloning serum supplementation are compared to a previously approved Opti MEM I Reduced Serum Medium control GIBCO cell culture liquid products are prepared by an aseptic process for which each step has been validated to ensure that all products meet the industry standard sterility assurance level of 107 i e product that demonstrates a contamination level of no more than 1 of 1000 units during the manufacturing process The highest level of sterility assurance equal to or greater than 10 cannot be achieved without terminal sterilization which is harmful to the performance of cell culture products 25 Accessory Products BioModule Units Additional BioModule Units that can be used for validation experiments are available separately from Invitrogen Ordering information is provided below For more information visit our web site at www invitroge
24. he product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Use of this product in conjunction with methods for the introduction of RNA molecules into cells may require licenses to one or more patents or patent applications Users of these products should determine if any licenses are required Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned by Invitrogen and claiming this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 This product and or its use may be covered by o
25. ing reagents and solutions Use the BLOCK iT Fluorescent Oligo with Lipofectamine 2000 Reagent for delivery of Stealth RNAi or siRNA to mammalian cells Follow the guidelines below when transfecting the BLOCK iT Fluorescent Oligo TM e The amount of BLOCK iT Fluorescent Oligo to use depends on the growth rate and transfection efficiency of the mammalian cells If you are transfecting your mammalian cell line for the first time we recommend evaluating several concentrations of lipid and varying the final concentration of the BLOCK iT Fluorescent Oligo from 10 to 200 nM to determine the optimal amount of TM BLOCK iT Fluorescent Oligo to use to obtain a strong fluorescence signal Note For most cell lines tested e g HEK293 A549 HeLa we obtain a readily detectable fluorescence signal when using 100 nM BLOCK iT Fluorescent Oligo for transfection e Prepare lipid BLOCK iT Fluorescent Oligo complexes as directed on page TM 17 Always dilute the BLOCK iT Fluorescent Oligo immediately before transfection i e do not store diluted Oligo into Opti MEM I Reduced Serum Medium Continued on next page 15 Transfecting Cells Continued Amount of RNAi to The amount of Stealth RNAi or siRNA duplex BLOCK iT Fluorescent Oligo Transfect Materials Needed 16 or the Stealth RNAi Negative Control Duplex required to achieve optimal target gene knockdown or minimal knockdown
26. invitrogen BioModule Transfection and Control Unit with BLOCK iT Technology For delivery of Stealth RNAi or siRNA into mammalian cells for RNAi analysis Catalog no WFGE06 Version A 6 December 2005 25 0882 ii Table of Contents Table OF COmtent commit on eines lr iii Experienced Users Procedure cccccccccsesessssssssesssesescecesessesesesesnsnsnesesesesescecseeuesesesesesesnansnesessseseeeeeeensienesesesanans v Kit Contents and Storage maene tienes tia nr Eana aa o aeae E aae Aa no na aa Ta EEOAE ora a ieai Eai vi Intro dU CIO 00 1 Overview aean hit ids hanes eines a it 1 Description Of COMPONENUS cccccceceesesesssnseseseseececeeeenesesesesesnsnsneseseseseeceeeenessseseeesesenesesesesuansnenesesesesceceeeananey 5 Experimental Overview 0 A Nise feat esata Atlas E 8 MethodS sei 10 General Guidelines inicia ia 10 Transhecting Cell AAA NS 14 Expected Results io fish testis sao bah Cased deis cid iodo 20 TOUDLESMO OUI Ess O 22 APPendNG nadan 24 lt AAA A A A RNA 24 Product Qualification 2 22 c5 fsice Sch eect a Lisshaend baka dense eee hi ehaeer desde ac ek aia aaia tel 25 Accessory Prod UCSI a aR Se ed anne e Beas a Mein eda Bats 26 Purchaser Noticas detente Sdn dees een Ad A Ad A dues Sete ctaees 28 RECESO RR 30 iv Experienced Users Procedure TM Introduction A brief experienced user s procedure for transfecting Stealth RNAi or siRNA molecules into mammalian cells using Lipofectamine 2000 Re
27. lth RNAi or standard siRNA into your mammalian cell line of interest for RNAi analysis TM e Include the BLOCK iT Fluorescent Oligo in your RNAi experiments to help you optimize your transfection conditions and to assess transfection efficiency in your mammalian cell line Once you have optimized your transfection conditions include the BLOCK iT Fluorescent Oligo in every RNAi experiment as an indicator of transfection efficiency e If you are transfecting RNAi molecules into a mammalian cell line of interest for the first time use the reagents provided to help you optimize your transfection conditions 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 to elicit gene silencing similar to traditional siRNA See next page for a description of the RNAi pathway TM Using Stealth RNAi for RNAi analysis offers the following advantages e Obtain effective target gene knockdown at levels that are equivalent to or greater than those achieved with traditional siRNA e Reduces non specific effects caused by induction of cellular stress response pathways e Exhibits enhanced stability for greater flexibility in RNAi analysis TM A large variety of ready to o
28. n com or call Technical Service page 24 Product Amount Catalog no BioModule Western Analysis Unit for chromogenic detection 20 transfers WFGE09 for chemiluminescent detection 20 transfers WFGE10 BioModule qRT PCR Unit 100 reactions WFGE01 1000 reactions WFGE02 BioModule Immunohistochemical Staining IHC Unit for Tissue 150 slides WFGE11 BioModule BLOCK iT Unit with Pol I miR RNAi Expression 20 reactions WFGE07 Vector BioModule BLOCK iT Unit with Lentiviral Pol II miR RNAi 20 reactions WFGE08 Expression System BioModule Microarray Unit with indirect labeling 15 arrays WFGE03 with direct labeing 15 arrays WFGE04 Additional Products Additional reagents that may be used with the BioModule Transfection and Control Unit are available separately from Invitrogen Ordering information is provided below For more information visit our web site at www invitrogen com or call Technical Service page 24 Product Amount Catalog no BLOCK iT RNAi Target Screening System w lacZ reporter 20 reactions K4916 00 Lipofectamine 2000 Reagent 0 75 ml 11668 027 1 5 ml 11668 019 Stealth RNAi Negative Control Kit 1 kit 12935 100 Stealth RNAi Negative Control Low GC 250 pl 12935 200 Stealth RNAi Negative Control High GC 250 pl 12935 400 Opti MEM I Reduced Serum Medium 100 ml 31985 062 500 ml 31985 070 CyQUANT Cell Prolifera
29. nd S M Bernstein E Beach D and Hannon G J 2000 An RNA Directed Nuclease Mediates Genetic Interference in Caenorhabditis elegans Nature 404 293 296 Hannon G J 2002 RNA Interference Nature 418 244 251 Jones A L Thomas C L and Maule A J 1998 De novo Methylation and Co Suppression Induced by a Cytoplasmically Replicating Plant RNA Virus EMBO J 17 6385 6393 Ketting R F Fischer S E Bernstein E Sijen T Hannon G J and Plasterk R H 2001 Dicer Functions in RNA Interference and in Synthesis of Small RNA Involved in Developmental Timing in C elegans Genes Dev 15 2654 2659 Li W X and Ding S W 2001 Viral Suppressors of RNA Silencing Curr Opin Biotechnol 12 150 154 Napoli C Lemieux C and Jorgensen R 1990 Introduction of a Chalcone Synthase Gene into Petunia Results in Reversible Co Suppression of Homologous Genes in trans Plant Cell 2 279 289 Nykanen A Haley B and Zamore P D 2001 ATP Requirements and Small Interfering RNA Structure in the RNA Interference Pathway Cell 107 309 321 Plasterk R H A and Ketting R F 2000 The Silence of the Genes Curr Opin Genet Dev 10 562 567 Romano N and Macino G 1992 Quelling Transient Inactivation of Gene Expression in Neurospora crassa by Transformation with Homologous Sequences Mol Microbiol 6 3343 3353 Continued on next page 30 References Continued Smith C J Watson C F
30. nd make sure that cells are healthy and greater than 90 viable before transfection e Maintain the same seeding conditions between experiments e Transfect cells at 30 50 confluence Transfecting cells at a lower density allows a longer time interval to elapse between transfection and assay time and minimizes the loss of cell viability due to cell overgrowth Depending on the nature of the target gene transfecting cells at higher densities may be suitable with optimization of conditions e To increase accuracy and reduce assay variability we recommend performing triplicate transfections for each sample condition TM e We recommend assessing BLOCK iT Fluorescent Oligo uptake at 6 24 hours post transfection however assay target gene knockdown levels following Stealth RNAi or siRNA delivery at a minimum of 24 72 hours following transfection You may also assay for target gene knockdown at various time points such as 24 48 72 96 hours post transfection TM e Determine the appropriate amount of BLOCK iT Fluorescent Oligo to use such that fluorescence signal is readily detectable For recommended reagent amounts to use see page 17 Note Once you have determined optimal transfection conditions using the BLOCK iT Fluorescent Oligo you may use these conditions as a starting point to transfect Stealth RNAi or siRNA with optimization as necessary TM e Test serum free media for compatibility with Lipofectamine
31. ndicator e Some reagents in the unit may be provided in excess of the amount needed Note e Individual documentation detailing general use are included with some of the products supplied in the BioModule Transfection and Control Unit To use the products specifically with the BioModule Transfection and Control Unit follow the recommended protocols in this manual vi Overview Introduction BioModule Units for Gene Expression Profiling Introduction The BioModule Transfection and Control Unit with BLOCK iT Technology provides qualified reagents and validated protocols to deliver Stealth RNAi see page 3 for details or siRNA short interfering RNA molecules into mammalian cells for RNA interference RNAi studies The BioModule Transfection and Control Unit provides reagents and controls required to perform transfection and assess transfection efficiency for RNAi analysis in mammalian cell lines The BLOCK iT Technology is a next generation RNAi technology employing synthetic Stealth RNAi or expression vectors containing an RNAi cassette to perform RNAi studies in mammalian cells A variety of RNAi products with BLOCK iT Technology are available from Invitrogen to facilitate RNAi analysis For more information visit the RNAi Central portal at www invitrogen com rnai or contact Technical Service page 24 The BioModule Transfection and Control Unit with BLOCK iT Technology is
32. ne or more of U S Patent No 6 506 559 and or foreign equivalents and is sold under license to Invitrogen Corporation by the Carnegie Institution of Washington 1530 P Street N W Washington DC 20005 A separate license from the Carnegie Institution of Washington may be required to use this product Continued on next page Purchaser Notification Continued Limited Use Label License No 196 Stealth RNAi The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or i
33. nt Oligo and Stealth RNAi or standard siRNA oligomers 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 e Highest transfection efficiency in many cell types and formats e g 96 well Refer to the Cell Lines database at www invitrogen com for a list of cell types successfully transfected TM e Nucleic acid Lipofectamine 2000 complexes can be added directly to cells in culture medium in the presence or absence of serum e Itis not necessary to remove complexes or change add medium after transfection but complexes may be removed after 4 6 hours Stealth RNAi Negative Control Duplex Medium GC is ideal for use in RNA interference RNAi experiments as a control for sequence independent effects following Stealth RNAi delivery in any vertebrate cell line The 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 Duplex Medium GC contains 48 GC content and is suitable for use with Stealth RNAi duplexes containing 45 55 GC The Stealth RNAi Negative Control Duplex is supplied in a ready to use format The BLOCK iT Flu
34. orescent Oligo is a fluorescein labeled dsRNA oligomer designed for use in RNAi analysis to facilitate assessment and optimization of cationic lipid mediated delivery or electroporation of dsRNA oligonucleotides into mammalian cells Using the BLOCK iT Fluorescent Oligo in RNAi studies offers the following advantages e Provides a good indication of the transfection efficiency with Invitrogen s Stealth RNAi standard unmodified siRNA or purified Dicer generated siRNA e Allows strong easy fluorescence based indication of transfection efficiency in every RNAi experiment Continued on next page Description of Components Continued Characteristics of the BLOCK iT Fluorescent Oligo The BLOCK iT Fluorescent 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 and growth rate of the cells TM The sequence of the BLOCK iT Fluorescent Oligo is not homologous to any known gene ensuring against induction of non specifi
35. rder pre designed Stealth RNAi products are available from Invitrogen to facilitate RNAi analysis page 27 or you may design and order custom Stealth RNAi for any target gene of interest using the BLOCK TM iT RNAi Designer from Invitrogen www invitrogen com rnaidesigner Continued on next page Overview Continued RNAi Pathway Purpose of this Manual 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 post transcriptional 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 amp Macino 1997 Cogoni amp Macino 1999 Romano amp 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 amp 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 RI
36. ses the observed express the target gene target gene siRNA was not active Design a siRNA to a different target region or convert the siRNA to a Stealth RNAi molecule and repeat the RNAi analysis Cytotoxic effects Transfection of siRNA activates e Convert the siRNA to a Stealth RNAi observed after toxic pathways that result in cell molecule and repeat the RNAi analysis transfection death e Test another target sequence to the same gene to confirm the toxicity response TM Lipofectamine 2000 Reagent may have cytotoxic effects Optimize the transfection conditions for your cell line by decreasing or varying the amount of Lipofectamine 2000 Reagent used Use the BLOCK iT Fluorescent Oligo to help you optimize transfection conditions for your cell line RNAi molecule targets an essential gene Reduce the amount of RNAi molecule transfected Note that doing so may negatively impact the level of knockdown observed Non specific effects siRNA targets regions with homology to other genes Convert the siRNA to a Stealth RNAi molecule and repeat the RNAi analysis No fluorescence signal detected with BLOCK iT Fluorescent Oligo Incorrect filters used to detect fluorescence Be sure to use the recommended filter sets for detection of fluorescence page 19 and use an inverted fluorescence microscope for analysis If desired allow the protein expression to continue for additional days
37. t you can use a range from 20 100 nM Stealth RNAi for 24 well format e Depending on the nature of the target gene transfecting cells at higher densities may also be considered when optimizing conditions e Note that for RNAi molecules inducing gt 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 To transfect cells in different tissue culture formats vary the amounts of Lipofectamine 2000 nucleic acid cells and medium used in proportion to the relative surface area as shown in the table With automated high throughput systems a complexing volume of 50 pl is recommended for transfections in 96 well plates Tip 20 uM Stealth RNAi or siRNA 20 pmol ul Note You may perform rapid 96 well plate transfections by plating cells directly into the transfection mix Prepare complexes in the plate and directly add cells at twice the cell density as in the basic protocol in a 100 ul volume Cells will adhere as usual in the presence of complexes Culture vessel Surface Area Volume of RNAi pmol in media Lipofectamine 2000 pl per well plating medium volume ul in media volume pl 96 well 0 3 cm 100 ul 5 pmol in 25 pl 0 25 plin 25 pl 24 well 2 cm 500 pl 20 pmol in 50 pl 1 0 pl in 50 pl 12 well 4 cm 1 ml 40 pmol in 100 ul 2 0 pl in 100 pl 6 well 10 cm
38. ted Results Continued BLOCK iT Fluorescent Oligo Results An example of results obtained after transfection of BLOCK iT Fluorescent Oligo into A549 cells are shown below A549 cells were plated at 8 x 10 cells well in a 12 well plate The cells were transfected with 100 nM BLOCK iT Fluorescent Oligo using 2 pl Lipofectamine 2000 Reagent as described in this manual Cells were visualized by fluorescence microscopy at 24 hours post transfection using the appropriate filters as described in this manual Fluorescence microscopy results are shown below and demonstrate the presence of BLOCK iT Fluorescent Oligo in virtually all cells indicating good transfection efficiency Fluorescent Brightfield 21 Troubleshooting Introduction Review the information in this section to troubleshoot your transfection and TM knockdown experiments with the BioModule Transfection and Control Unit Problem Reason Solution Low knockdown of the target gene observed e Low transfection efficiency e Expression assay not performed correctly See below e Be sure the expression assay was performed correctly See page 12 for guidelines Low levels of gene knockdown observed due to low transfection efficiency e Antibiotics added to the media during transfection Cells too sparse at the time of transfection e Not enough RNAi duplex transfected e Not enough transfection reagent used
39. the page to download the MSDS for your product Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Service Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Service Representatives Invitrogen assumes no responsibility or liability for any special incidental in
40. these factors into account when designing your RNAi experiments TM To validate your Stealth RNAi or siRNA you must measure the effect of the RNAi molecule 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 e Cell proliferation assays using the CVQUANT Cell Proliferation Assay Kit page 26 e Quantitative RT PCR qRT PCR e Signal transduction pathway analysis using CellSensor Cell Lines page 27 e Reporter based target screening system such as the BLOCK iT RNAi Target Screening System with lacZ reporter page 26 e Protein expression assay using enzyme activity immunocytochemistry immunohistochemistry western blotting or ELISA Prior to performing RNAi experiments 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 e High sensitivity e Low background e Signal stability e Fast efficient easy to perform If you are measuring protein levels to analyze the Stealth RNAi or siRNA 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
41. tion Assay Kit 1 kit C7026 26 Continued on next page Accessory Products Continued BLOCK iT RNAi Designer BLOCK iT RNAi Products CellSensor Cell Lines Antibodies The BLOCK iT RNAi Designer is an online tool www invitrogen com rnaidesigner to help you design and order Stealth RNAi or siRNA molecules for any target gene of interest The RNAi Designer incorporates published rules on RNAi design into a proprietary algorithm to design most effective RNAi sequences to obtain high level gene knockdown TM A large variety of BLOCK iT RNAi products are available from Invitrogen to facilitate RNAi analysis including Stealth RNAi the Validated Stealth RNAi Stealth RNAi Collection and a large selection of RNAi vectors For details visit the RNAi Central portal at www invitrogen com rnai or contact Technical Service see page 24 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 quali
42. transfection efficiency in RNAi experiments with Stealth RNA or siRNA Stealth RNAi Negative Control Duplex Medium GC for use as a negative control for the RNAi response Opti MEM I Reduced Serum Medium is a multi purpose proven medium used for diluting the Lipofectamine 2000 Reagent and nucleic acid during transfection The medium is also useful in reducing serum requirements for a wide variety of cell lines and applications and has been effective in the growth and maintenance of adherent and non adherent cell lines 1X RNA Annealing Dilution Buffer for use in diluting RNAi molecules for transfection if necessary For more information about each component see page 5 TM To use the BioModule Transfection and Control Unit you will TM Obtain synthetic Stealth RNAi or siRNA duplex to the target gene of interest Transfect mammalian cell line of choice with the Stealth RNAi or siRNA using Lipofectamine 2000 Reagent and Opti MEM I Reduced Serum Medium Assay for gene knockdown using an assay of choice Continued on next page Overview Continued Uses for the BioModule Transfection and Control Unit Stealth RNAi Use the BioModule Transfection and Control Unit with BLOCK iT Technology in your RNAi experiments for the following purposes TM e Use Lipofectamine 2000 Reagent for highly efficient delivery of dsRNA oligomers including the BLOCK iT Fluorescent Oligo and Stea
43. ts components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned by Invitrogen and claiming this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 29 References Ambros V 2001 MicroRNAs Tiny Regulators with Great Potential Cell 107 823 826 Anandalakshmi R Pruss G J Ge X Marathe R Mallory A C Smith T H and Vance V B 1998 A Viral Suppressor of Gene Silencing in Plants Proc Natl Acad Sci USA 95 1307
44. ty antibodies including the Zymed Antibodies is available from Invitrogen for use in Western immunodetection immunohistochemistry or ELISA assays For details visit www invitrogen com or contact Technical Service page 24 27 Purchaser Notification Limited Use Label License No 27 Lipofectamine 2000 Reagent Limited Use Label License No 173 Inhibition of Gene Expression by Double stranded RNA 28 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a to not transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of t
45. ute 1 ul in 50 ul Opti MEM I Reduced Serum Medium Mix gently and incubate for 5 minutes at room temperature Note Combine diluted Lipofectamine 2000 with diluted RNA within 30 minutes d After the 5 minute incubation combine the diluted oligomer with the diluted Lipofectamine 2000 total volume is 100 ul Mix gently and incubate for 20 minutes at room temperature solution may appear cloudy TM Add the oligomer Lipofectamine 2000 complexes to each well containing cells and medium Mix gently by rocking the plate back and forth Incubate the cells at 37 C in a CO incubator for 24 96 hours until you are ready to assay for target gene knockdown Assess BLOCK iT Fluorescent Oligo uptake at 6 24 hours post transfection Medium may be changed after 4 6 hours An example of gene knockdown results are shown on page 20 Continued on next page 17 Transfecting Cells Continued Optimizing Transfection Scaling Up or Down Transfections To obtain the highest transfection efficiency and low non specific effects optimize transfection conditions by varying RNA and Lipofectamine 2000 Reagent concentrations TM e 1phlLipofectamine 2000 Reagent per well is recommended as a starting point but optimization of transfection conditions may be required A range of 0 5 ul to 1 5 ul Lipofectamine 2000 Reagent is recommended for 24 well format e 40nM Stealth RNAi is recommended as a starting point bu
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