Baec, bovine aortic endothelial cells, sirna, lipofectamine rnaimax, 778. Jan 18, 2011 primary hepatocytes are the best resource for in vitro studies directed at understanding hepatic processes at the cellular and molecular levels, necessary for novel drug development to treat highly prevalent diseases such as nonalcoholic steatohepatitis, cardiovascular disease and type 2 diabetes. Shared reagents dna transfection sirna transfection growth medium optimem medium for complexing dna p3000 reagent lipofectamine 3000 reagent sirna lipofectamine 3000 reagent 96well 0. Differentiated thp1 cells were treated with 60 nm sirna. Invitrogen lipofectamine rnaimax transfection reagent 0. Invitrogen lipofectamine 3000 transfection reagent. Lipofectamine 2000 transfection protocol 10cm plates reagents. Whole field 20x magnification view of gfp expression in raw 264. Prewarm 50ml of optimum stored in cold room at 4c 10min. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. Incubate the cells at 37c in a co2 incubator for 2496 hours until you are ready to assay for gene knockdown. On the other hand, dendrimer mh47, which aggregated and bound. Photograph of expected results dna lipofectamine scaling up or down. So i asked one of my lab mates and he told me to use just 1ul.
I usually use 56 ulwell lipofectamine 2000 for an sirna concentration of 150 nm in a 24well plate as suggested by the vendor. Nucleic acidlipofectamine2000 complexes can be added directly to cells in culture medium, in the presence or absence of serumantibiotic. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Product is guaranteed for 6 months from the date of shipment if stored properly.
After a 5 minute incubation in optimem, the lipofectamine 2000 and dna are combined and incubated for 20 minutes with gentle shaking at room temperature. 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. Jul 24, 2007 lipofectamine 2000 has been used successfully to transfect short interfering rnas sirna into mammalian cells for rna interference rnai studies gitlin et al. Description lipofectamine2000 transfection reagent is a proprietary formulation for transfecting nucleic acids dna, rna, and mrna into a wide range of eukaryotic cells.
Transfection efficiency determined by famlabeled control sirna. Before seeding cells for virus package, make sure hek293t cells are in rapid replication state by growth cells to approximately 75%80% confluence in a 10 cm cell culture dish and passaging at 1. 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. Can you suggest a protocol for transfecting c2c12 myotubes not. Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Dilute sirnachimera with optimem i reduced serum medium invitrogen up to 50 l and vortex for a few seconds dilute appropriate amount of lipofectamine 2000 by optimem i reduced serum medium to 50 l and vortex for a few seconds for siha and skov3 cells, dilute 1. For the cell lines easy to transfection, you can use either of the procedures reverse or forward.
Jun 10, 2011 lipofectamine 2000 is commonly used for sirna transfections. Lipofectamine 2000 has been used successfully to transfect short interfering rnas sirna into mammalian cells for rna interference rnai studies gitlin et al. Although this amount is good for a wide range of sirna concentrations, if more sirna is transfected, the amount should be increased accordingly. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. The client service offered by sitools is excellent.
Lipofectamine 2000 reagent thermo fisher scientific. As sirna itself had no obvious cytotoxic effect on bel7402 cells, the cytotoxicity of mnhap sirna and lipofectamine 2000 sirna complexes were quite possibly due to mnhap and lipofectamine 2000 themselves. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Effects of lipofectamine 2000 sirna complexes on autophagy in hepatoma cells. On the day of transfection, complexes are prepared in a multiwell plate and incubated for the recommended complex formation time. Comparison of small interfering rna sirna delivery into bovine. Inspired by a recent report of sirna transfection with a 22residue. I want to try to use hela cell and lipofectamine 2000 to. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Dt40, chicken bursa, sirna, lipofectamine rnaimax, 778075 778150 page. Comparative nucleic acid transfection efficacy in primary. 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. The protocal is from thermo fisher scientific important guidelines for transfection 1. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells.
However, few studies have examined cellular responses to this delivery system. Ntersirna nanoparticlemediated knockdown of gene expression xtremegene 360 transfection reagent if additional help is needed, please email our technical services group. When i transfected sirna with lipofectamine with the amounts that appear in the protocol, all my cells died. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types.
Pool dishes of cells that have been prepared for transfection. Advanced transfection with lipofectamine 2000 reagent. After the 5min incubation, combine the diluted sirna with the diluted lipofectamine 2000 total volume is 100. Full text effective small interfering rna delivery in. Combine the diluted sirna with the diluted lipofectamine 2000, mix gently, and incubate for 20 min at room temperature.
Alexa fluor488 for 5 hours and cellular uptake is assessed after 24 hours by flow cytometry and confocal microscopy. Highly efficient sirna delivery into primary cultures of rat. Stereoselective ph responsive peptide dendrimers for sirna. I would suggest the modified lipofectamine 2000 invitrogen protocol described. Lipofectamine 3000 reagent is suitable for novel genome editing applications. There is a need to identify simple methods to genetically manipulate primary hepatocytes and. Moreover, xenograft tumor models demonstrated that specific gene knockdown is sustained, i. Preadipocytes were seeded in 6well plates, and sirna transfection was carried out 2 days after the confluence of preadipocytes. According to the quantitative analysis shown in fig. Transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Nter sirna nanoparticlemediated knockdown of gene expression xtremegene 360 transfection reagent if additional help is needed, please email our technical services group.
Rubi fructus rubus coreanus inhibits differentiation to. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Stealthsirna transfection protocol lipofectamine 2000. In addition, optimized sirna delivery into bmdm by electroporation achieved. Ive been working with sirnas just for a couple of weeks and i have more doubts that answers. Abstract lipofectamine 2000 lipo2000 delivery system is commonly used for short interfering rna sirna transfection, whereas the cellular. Transfection protocols are used in biological laboratories for introduction of foreign plasmid dna, small therapeutic rna, or protein molecules into cells of a eukaryotic nature. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Effective small interfering rna delivery in vitro via a new stearylated cationic peptide baoling chen,1,2 ran pan,1,2 diana askhatova,1 p chen1,21department of chemical engineering, 2waterloo institute for nanotechnology, university of waterloo, waterloo, on, canadaabstract. I want to try to use hela cell and lipofectamine 2000 to do. Rnai or technical service, as are celltype specific rnai transfection protocols see rnai protocols. Thee two most important factors in choosing transfecting reagent are. Lipofectamine 2000 transfection reagent from invitrogen. How does transfection with lipofectamine 2000 work.
The presence of optimem resulted in significantly higher transfection rates when lipofectamine 2000 was used. Lipofectamine 2000 kinda sucks, to be honest, that might be the source of the problem. Even so, mnhap was less toxic than lipofectamine 2000 as demonstrated in fig. Lipofectamine 2000sirna complexes cause endoplasmic reticulum. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone. Freshly passaged cells are then directly added to the multiwell plates containing the transfection complexes at twice the density of a standard forward trans. The intracellular trafficking mechanism of lipofectamine. The vector contained an ofp reporter gene and was transfected. Scaling up or down lipofectamine 3000 reagent transfections. L sirna 10 pmol were individually diluted and incubated in 250. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. Add 6l of lipofectamine2000 from biostores and 100 l of.
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. The comparative utility of viromer red and lipofectamine. They adapted a system previously used to transfect plant cells to myotubes from chicken and c2c12 myotubes. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with different amounts of lipofectamine 2000 5ul, 10ul in the 6well plate. This active endosome escape technology is safe and maximizes transfection efficiency as it is using a natural uptake pathway. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Lipofectamine 2000, lipofectamine rnaimax and dharmafect 3. Cellspecific transfection protocols thermo fisher scientific us. Lipofectamine 2000 transfection reagent dartmouth college. Highly efficient sirna delivery into primary cultures of. Has anyone tried sirna transfection with lipofectamine3000. Rnai in cultured mammalian cells using synthetic sirnas. Despite this, a satisfactory mechanismbased explanation of their. The in vitro transfection studies on bel7402 cells revealed that the proportion of fampositive cells increased remarkably following transfection with mnhapsirna and lipofectamine 2000sirna complexes compared to that with sirna alone.
Transfection protocol lipofectamine 3000 transfection. Mix gently and incubate for 20 min at room temperature to allow the sirna. This reference provides general guidelines and procedures to transfect sirna into mammalian cells using lipofectamine 2000. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Effects of lipofectamine 2000sirna complexes on autophagy. Sendai virus was propagated in 10 to 11dold chicken eggs that were inoculated. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100. The introduction of genes into glial cells for mechanistic studies of cell function and as a therapeutic for gene delivery is an expanding field. L optimem medium invitrogen, carlsbad, ca, usa for 20 min. A crucial bottleneck in rna interferencebased gene therapy is the lack of safe and efficient delivery systems. Lipofectamine 2000 is commonly used for sirna transfections. A day 1 sitnfr1 sishp1 sigfp normoxia 20% o 2 hypoxia 1% o 2 day 2. Add the oligomerlipofectamine 2000 complexes from step 2. It increases the likelihood of successful cleavage and recombination with talens or crisprs through highly efficient transfection, ultimately maximizing the efficiency of genetic modifications and simplifying the downstream processes.
Lipofectamine 2000 procedure for simultaneously transfecting. 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. Cotransfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Sep, 2005 both lipofectamine 2000 and dna are mixed with optimem media and the quantity of each is dependant on the type of culture vessel i. Effects of lipofectamine 2000sirna complexes on autophagy in. I use 24well plates for transfections in triplicates. Both lipofectamine 2000 and dna are mixed with optimem media and the quantity of each is dependant on the type of culture vessel i. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines.
Transfection of adherent cells with sirnamirna traditional protocol. Lipofectamine 2000 may be used in the following applications. Though viral vector based systems do exhibit good delivery efficiency and longterm production of the transgene, the need for transient gene expression, broad and rapid gene setup methodologies, and safety concerns regarding in vivo 0. I have used both lipofectamine 2000 and 3000 and always get the same results. For each transfection sample, prepare oligomerlipofectamine 2000 complexes as follows. Resuspend the sirna in h 2 o to a concentration of 100. Geneporter 3000 lipofectamine 2000 transfection reagent bgal expression, pgwell figure 2. 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. Troubleshooting for transfection of the crisprcas9 using. Highest transfection efficiency in many cell types and formats e. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. I would like to transfect 293t cells with lipofectamine 3000. Add the oligomerlipofectamine 2000 complexes to each well containing cells and medium. I recommend using lipofectamine rnaimax, because you can do the transfections without removing the antibiotics from the medium prior to it.
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