Effect of clic1 gene silencing on proliferation, migration. Bioreducible cationic polymerbased nanoparticles for. For example, for co transfection of plasmid dna and sirna in a 24well plate, we would recommend using. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. This application note describes optimizing the reverse transfection of synthetic guide rna into a cas9expressing cell line using dharmafect transfection reagents read note knockout protocol. Guidelines for transfection of mammalian cultured cells. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. In reverse transfections, the complexes are prepared inside the wells, after which cells and medium are added. The cells are transfected when they reach 80% confluence. Because the cells and sirna reagent complexes are prepared on the same day, we recommend using 2. For reverse transfection, cells in optimal growth phase are prepared so that it is possible to seed 2. In solidphase rev transfection, sirna complexes using cationic liposomes such as commercially available lipofectamine 2000 invitrogen, carlsbad, ca, usa and lipofectamine rnaimax invitrogen are attached to the bottom of cell culture plates by freezedrying 4 8 9, and cells take up these complexes after plating on the culture plate. Stealthsirna transfection protocol lipofectamine 2000. Rnaimax forward transfections lipofectamine thermo.
Rnaimax reverse transfections lipofectamine thermo fisher. Reverse transfection of cells on ment is reduced owing to the small amount of sirna needed plasmid or sirna arrays5. The dharmafect volumes and sirna amounts for reverse transfection are usually lower than the amounts needed for traditional transfection. Realtime rtpcr analysis was performed to examine akt3 from rna extracted from mcf7adr cells transfected with. With proprietary shaved cell transfection protocol, polyjet left panel gives up to 70% gfp positive cells vs. I tried a pool of 3 sirna transfection in hela cells using rnaimax but all of of them failed. According to the instructions, sirna lipofectamine 2000 was used for transfecting gbc cells. I am trying to transfect a luciferase reporter construct. Incubate 5 min at room temp then mix lipofectamine and sirna dilutions. Jun 01, 2004 knockdown of lamin ac protein levels, using the same sirna, can also be accomplished by transfection using lipofectamine 2000. Realtime rtpcr analysis of akt3 gene by transfection of sirna in mcf7adr cells. Dharmafect transfection reagentssirna transfection.
After transcription, a stemloopstem shrna molecule is produced. Seven steps toward rnai success thermo fisher scientific. The vector contained an ofp reporter gene and was transfected with lipofectamine 2000 or lipofectamine 3000 reagent into a u2os and b hepg2 cell lines. Co transfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0.
All knockdown values are normalized to scrambled control rna. For researchers interested in other delivery methods, we also offer additional invitrogen lipofectamine reagents for mrna transfection and lentiviral production as well as the invitrogen neon transfection system for. If you want to use lipofectamine rnaimax for your cotransfections, perform a reverse transfection as described on page 2 with the following. Lipofectamine 2000 reagent is supplied and should be stored as follows. Optimization of cellbased cdna microarray conditions for. Reverse transfection on cell arrays for high content. Dotap functionalizing singlewalled carbon nanotubes as non. Can anyone suggest whether transfection medium must be. Stealth rnai, sirna, or diced sirna into a broad range of mammalian cell lines. Evaluation of small interfering rna delivery into cells by. Rnaimax reverse transfections lipofectamine thermo. Exceptional broad spectrum transfection cutting edge delivery of plasmid dna andor small rnas sirna, mirna, and crispr guide rna outperforms lipofectamine 2000 in 28 of 41 tested cell lines. Perform reverse transfection by preparing the sirna transfection complexes inside the wells, and then adding cells and medium. I am trying to transfect a luciferase reporter construct into.
Lipofectamine 2000, and lipofectamine 2000 cd x broad spectrum dna deliveryachieve high expression in many cell types, including hardtotransfect cells and insect cells x animal origin freetransit2020 provides high performance with maximum compatibility description transit2020 reagent is a new and versatile transfection solution for. Reverse transfection of cells on plasmid or sirna arrays 5,6,7,8,9,10 is a powerful alternative method to perform highthroughput transfections for phenotypic data acquisition by light microscopy. Reverse transfection an overview sciencedirect topics. Conventional liquidbased transfection with liposomes, such as lipofectamine 2000, requires 30 times the volume of transfection mixture as that used in our method. Use the procedure on below to transfect cells with plasmid dna. Reverse transfection of cells on plasmid or sirna arrays is a powerful alternative method to. Scaling up or down lipofectamine 3000 reagent transfections. Transfection was performed as described in protocol 2 with the following modifications. Moreover, after interaction with swntdotap, sirna of human telomerase reverse transcriptase was bound, condensed and stabilized. Dotap functionalizing singlewalled carbon nanotubes as. Transfection efficiency and protein expression using geneart crispr nuclease vector. This molecule is processed by the dicer enzyme to generate a doublestranded sirna effector. I have used sirna knockdown in primary cells huvec and cell lines as 293t. Lipofectamine 2000 reagent the blockit rnai basic control kit human includes lipofectamine 2000 reagent box 2 for highefficiency transfection of dna and dsrna oligomers i.
Application note gene delivery solutions stem cell. Perform reverse transfection by preparing sirna or mirna transfection complexes inside the wells, and then adding cells and medium. Highly efficient reverse transfection with sirna in. Reverse transfection is the protocol of choice for most applications, as it frequently yields better transfection efficiency and facilitates the experimental protocol by combining cell seeding and transfection into a single step. Interestingly, we found that lipofectamine 2000, while less toxic towards cancer cells, was extremely cytotoxic using manufacturers recommended transfection condition towards nscs, which led to 95% cell death within 48 h of being transfected with the negative control sirna fig. Transfection efficiency varies according to the cell type and transfection. General guidelines for transfection follow these general guidelines when using lipofectamine 2000 to reverse transfect sirna or stealth rnai duplexes into mammalian cells. Introduction highthroughput parallel transfection techniques are a prerequisite to perform largescale experiments, such as genomewide rnai screens, in mammalian tissue culture cellss in such studies, transfections are typically performed in 96 or 384well plates using liquidhandling robotics. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0. Work flow for multiplexing sirna assays by solidphase.
Guide to packaging and transduction of target cells user manual. Guidelines for transfection use the procedure on below to transfect cells with short interfering rna sirna or invitrogen stealth rnai. If you want to use lipofectamine rnaimax for your cotransfections, perform a reverse transfection with the following modifications. Sirna and plasmid cotransfection using lipofectamine 3000. Lipofectamine 2000 reagent thermo fisher scientific. To optimize transfection conditions, determine the conditions that result in. Work flow for multiplexing sirna assays by solidphase reverse transfection in multiwell plates holger erfle,1,4 beate neumann, 2phill rogers, jutta bulkescher, jan ellenberg,3 and rainer pepperkok1 solidphase reverse transfection on cell microarrays is a highthroughput method for the parallel transfection of. Luciferase expression was compared at 24 hours post. Mar 01, 2016 next, we performed titration of sirna quantity 2. To optimize transfection conditions, determine the conditions that result in maximum gene silencing with minimal cytotoxicity. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Optimizing stealth rnai or sirna transfection to obtain the highest transfection efficiency and low nonspecific effects, optimize transfection conditions by varying rna and lipofectamine 2000 concentrations. Lipofectamine rnaimax for your cotransfections, perform a reverse transfection as described on page 2 with the following modifications.
The protocol indicated below is a reverse transfection protocol. Plate cells so they will be 7090% confluent at the time of transfection. Reverse transfection of stealth rnai or sirna using. Lipofectamine 3000 transfection reagent the preferred choice for transfection of two types of stem cells. For a detailed description of sbis lentivector expression system, please refer to the lentivector expression systems. M by reverse transfection time 24 h, 48 h, 72 h and confirmed 3. In my experience its better to replace transfection media with serum containing media after 34 h in case of primary cells.
Reverse transfection use this procedure to reverse transfect stealth rnai or sirna into mammalian cells in a 24well format for other formats, see scaling up or down transfections, page 3. For cotransfections of plasmid dna and stealth rnai or sirna into mammalian cells, we recommend using lipofectamine 2000 catalog no. Reverse arrows refer to the pcr primers contained in this product to confirm positive clones. Transitx2 transfection reagent enables superior transfection in a variety of cell types. Prepare plasmid dnalipid complexes recommend 2 doses of lipid.
Oct 01, 2007 however, after reverse transfection, the attached sirna was present inside cells and the efficiency of transfection was uniform in all regions of the well. Transitx2 dynamic delivery system and lipofectamine 2000 transfection reagent were used to transfect plasmid dna encoding luciferase into 41 different cell types at three reagenttodna ratios. I am using the standard protocol for lipofectamine rnaimax plate cells to 6080% confluency, dilute 9l lipofectamine in 150l optimem, dilute 30 pmol sirna in 150l optimem. Transfection and ancillary reagents horizon discovery. Cells are added directly to the lipofectamine 2000 rna complexes and transfection occurs while cells are attaching to the well. Highly efficient reverse transfection with sirna in multiple. A comparison showing transfection efficiency of polyjet reagent vs. Third, parallel cell seeding in a single chamber for 384 to perform highthroughput transfections for phenotypic data knockdown experiments with no physical separation between. Lipofectamine 2000 right panel around 5% efficiency. For high throughput sirna screening in easytotransfect cells, we recommend using a reverse transfection protocol see this reverse transfection protocol.
Advanced transfection with lipofectamine 2000 reagent. I did get the knockdown to work when using my sirna with lipofectamine 2000, however. Dharmafect transfection reagentssirna transfection protocol. B transfection results using 20 nm sirna with polymer concentrations ranging from 11. Nanotopographymediated reverse uptake for sirna delivery. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Jan 01, 2012 lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. Nanoru, on the other hand, was seen to be biocompatible. M sirna concentration and 48 h as the best condition.
When optimizi ng transfection conditions, follow the guidelines below. To ensure uniform uptake of stealth rna, make sure that cells are plated uniformly across the wells. Transfection amounts 96well 24well 6well final sirna used per well 1 pmol 5 pmol 25 pmol final lipofectamine rnaimaxused per well 0. Development of a celldefined sirna microarray for analysis. M was transferred to the 384well vbottom plate containing 2. Work flow for multiplexing sirna assays by solidphase reverse transfection in multiwell plates holger erfle,1,4 beate neumann, 2phill rogers, jutta bulkescher, jan ellenberg,3 and rainer pepperkok1 solidphase reverse transfection on cell microarrays is a highthroughput method for the parallel transfection of mammalian cells. Pdf reverse transfection on cell arrays for high content. We recommend gibco optimem i reduced serum medium cat. Validation of antibody specificity using reverse short. Guidelines for transfection of mammalian cultured cells the efficiency with which mammalian cells are transfected with sirna varies according to the cell type and transfection agent used. To transfect cells with sirna, follow the protocol as. The lipofectamine 2000 control shown used 20 nm sirna. Rnaimax forward transfections lipofectamine thermo fisher. For highly efficient transfection in a broad range of mammalian cell types, use lipofectamine 2000 reagent catalog no.
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