Xtremegene sirna transfection reagent polymer reagent for. This protocol can also be followed when cotransfecting plasmid dna and mirna by simply. I am trying to knockdown cmyc andor ras using mirna, let7. Review and cite lipofectamine protocol, troubleshooting and other. Advanced transfection with lipofectamine 2000 reagent. Transfection amounts per well use 10 nm mirna duplex as a starting point.
Prognostic implications of microrna100 and its functional roles in. I transfect my pc3 cell line with lipofectamine 2000 and my sirna plk1. A sample protocol is listed here for transfection experiments performed in 6well plates. This protocol can also be followed when cotransfecting plasmid dna and mirna by simply substituting mirna for sirna. By use of this product, you accept the terms and conditions outlined in the licensing and warranty statement.
Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Dharmafect transfection reagents provide efficient and reliable sirna transfection at low sirna concentrations with low cellular toxicity. The first aim of the current study was to evaluate the effect of mirna transfection on the global gene expression change in human granulosa cell line kgn. Genscript recommends using lipofectamine 2000 for all transfections. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. This reference provides general guidelines and a procedure to cotransfect plasmid dna and an rnai molecule i. Cotransfection protocol for plasmid dna and sirnamirna. The transfection efficiency of mir21 and lipofection protocol. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from a difficult to transfect cell line that had been giving us trouble with the lipofectamine 2000. Highest transfection efficiency in many cell types and formats e. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. We produce mirna lentiviral stocks in 293t cells using the transfection conditions summarized in a table below. Feb 06, 2010 i am trying to knockdown cmyc andor ras using mirna, let7. Lipofectamine 2000 transfection reagent from invitrogen.
The cloning site of premirna genomic fragments is within the intron of human ef1. Quantitative pcr following mirna mimic transfection or addition post lysis. For additional information on scaling your transfection reaction, see page 4. Can i use the lipefectamine 2000 for mimic mirna transfection. Plate an amount that will result in approximately 85 to 90% confluence see table below on the day of transfection. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. Thermo scientific dharmafect transfection reagents deliver specialized formulations for successful delivery of sirna to more cell lines. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2.
Jun 01, 2004 read advanced transfection with lipofectamine 2000 reagent. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Target prediction and validation of micrornas expressed from. Lipofectamine rnaimax reagent protocol thermo fisher scientific. Microrna7085p acts as a therapeutic agent against metastatic. Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell growth, investigation of gene regulatory elements, and to produce a specific protein. On measuring mirnas after transient transfection of mimics or. The following procedure is provided as an example only. Lentivectorbased microrna precursor constructs cat. Mir8 downregulates mirna processing in hela cells by. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. Can i use the lipefectamine2000 for mimic mirna transfection.
Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. A lipidbased transfection reagent, such as xtremegene sirna transfection reagent, can provide a convenient, reliable, and efficient vehicle for delivering sirnas into animal cells, enabling the study of cellular and functional consequences of gene knockdown. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types. On measuring mirnas after transient transfection of mimics or antisense inhibitors. Cells were seeded into plates 24 h before transfection. The mirna of interest can be delivered into cells by transient transfection of the plvmirna plasmid or lentiviral transduction while the mirna lentiviral stock is prepared from cotransfecting hek 293t. Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. The amount of ptk9 mrna was measured relative to 18s rrna levels using qrtpcr and then normalized to the mrna levels of the negative control, 48 hours posttransfection. The manufacturers of transfection reagents, the suppliers of lentiviral packaging constructs, and many academic laboratories have provided protocols for producing lentiviral stocks. Using lipofectaminez 2000 for transfection provides the following advantages. Optimizing transfection of mirna sirna, microrna and rnai.
The highest transfection efficiency in many cell types and formats e. 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. Invitrogen lipofectamine 2000 transfection reagent. Mar 27, 2007 lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Superior performance for co transfection of sirna and plasmid dna. So i asked one of my lab mates and he told me to use just 1ul lipofectamine for 20pmol of sirna thats the protocol specificationsbut instead for each well, use that amount for three wells. Lipofectamine 2000 transfection protocol 10cm plates reagents. Both lipofectamine 2000 and lipofectamine rnaimax reagents are cationiclipid transfection reagents formulated for superior transfection efficiencies on a variety of cell types. Sirna and plasmid cotransfection using lipofectamine 3000. The biomodule transfection and control unit includes. Lipofectaminez 2000 is a proprietary formulation suitable for the transfection of nucleic acids into eukaryotic cells. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Jan 24, 20 quantitative pcr following mirna mimic transfection or addition post lysis.
Read advanced transfection with lipofectamine 2000 reagent. T47d cells were transfected in a 12well plate using 3 l of transitx2 or lipofectamine 2000 and 50 nm mirna according to each manufacturers protocol. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells and provides the following advantages. Establishment of lipofection protocol for efficient mir21 transfection. Dysregulated micrornas in neurons could cause many nervous system diseases. Stealthsirna transfection protocol lipofectamine 2000. Lipofectamine 2000 reagent for highly efficient delivery of dsrna oligomers into a wide variety of mammalian cells blockit fluorescent oligo, a fluoresceinlabeled doublestranded rna dsrna oligomer for use as an indicator of transfection efficiency in rnai. Xtremegene hp dna transfection reagent is a proprietary blend of lipids and other components supplied in 80% ethanol, filtered through 0. B vector according to the manufacturers protocol invitrogen and then. The ability to alter microrna mirna abundance is crucial for studying mirna function. Lipofectamine 2000 dna transfection reagent protocol.
How to perform sirna transfection with lipofectamine rnaimax protocol. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Once reconstituted in nucleasefree water, the mirna is ready to transfect and can be used at your choice of final concentration. Hela, a549, panc1, mb231 total transfection volume. Cy3 sirna formed in optimem using lipofectamine 2000 at 2, 6 and 24 hours post transfection. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone has found an optimal lipo 3000 dna ratio for this cell line. Synthetic mirna mimics from dharmacon or shanghai genepharma were transfected into cells following the dharmafect1 transfection protocol t2001. See page 2 to view a typical rnaimax transfection procedure.
To investigate the influence of medium and transfection time, primary hepatocytes were transfected with metafecte pro, lipofectamine 2000 or targefect in the presence of dmem figure 2a or optimem, withwithout virofect enhancer, for 3 hours or 16 hours figure 2a, 2b, 2c. Dna complexes were added to cells that had already been allowed to attach to the plate b. Transfection of mir7085p mimics in high metastatic pg, a549 and h1299 cells induced cell. Transfection and genome engineering fisher scientific. On measuring mirnas after transient transfection of mimics. Lipofectamine rnaimax reagent is designed specifically for the delivery of sirna and mirna while lipofectamine 2000 reagent delivers dna or sirna with excellent. Ca were cotransfected into hek293t cells as previously described 3. Introducing the monster green fluorescent protein phmgfp vector using the transfast transfection reagent and the optimized transfection conditions resulted in the cell notes issue 7 2003 14 neuroscience transfecting a human neuroblastoma cell line with monster green fluorescent protein by natalie betz, ph. Dna complexes were added to wells so that transfection could occur as cells were attaching to the plate. For other tissue culture formats, refer to the user protocol.
Xtremegene hp dna transfection reagent sigmaaldrich. Lipofectamine 2000 is primarily used for delivery of plasmid dna, but it also works well for delivery of sirna and mirna mimics. Jul 24, 2007 lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of mammalian cell types in vitro using a simple protocol. The success of these manipulations is often assessed using qpcr, but this does not accurately report the level of. Superior performance for cotransfection of sirna and plasmid dna. General description short interfering rna sirna can be directly introduced into cells by transfection. Outlined below is an easytofollow cotransfection protocol for plasmid dna and sirna using transitx2 dynamic delivery system in a 6well format. The protocal is from thermo fisher scientific important guidelines for transfection 1.
Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Invitrogen lipofectamine rnaimax transfection reagent. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. For other tissue culture formats, refer to the user protocol pdf. I will try to transfect mirna to mcf7 cells but i have no transfection reagent like lipofectamine. Unlike for conventional dna transfection, transport of the nucleic acid into the cell nucleus is not necessary for rna transfection.
Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. 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. To achieve this there is widespread use of both exogenous doublestranded mirna mimics for transient overexpression, and single stranded antisense rnas antimirs for mirna inhibition. Rnai transfection protocol see page 2 to view guidelines for transfecting mirnas using lipofectamine rnaimax reagent.
Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects. Media were replaced with antibioticsfree media 612 h before transfection. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lipofectamine 2000 reagent thermo fisher scientific.
I use 24well plates for transfections in triplicates. Comparative nucleic acid transfection efficacy in primary. Lipofectamine 2000 reagent and lipofectamine ltx reagent. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Dec 02, 2015 transient transfection of mirna mimics. Biomodule transfection and control user manual unit with. Lipofectamine 2000 and lipofectamine rnaimax transfection. Lipofectamine 2000 is a cationic liposome based reagent that provides high transfection efficiency and high levels of transgene expression in a range of. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. When i transfected sirna with lipofectamine with the amounts that appear in the protocol, all my cells died. Stealth rnai, sirna, shrna plasmid or mirna plasmid into mammalian cells using lipofectamine 2000 reagent are described in this section. Optimized delivery of sirna into 3d tumor spheroid cultures. In some studies, the cationic lipofection reagents, like lipofectamine 2000, were efficient in. Thee two most important factors in choosing transfecting reagent are.