This is most often an endogenous proteincoding gene, although some noncoding rnas can also be silenced effectively. Scaling up or down lipofectamine 3000 reagent transfections. Lipofectamine is formulated to give high transfection efficiency with little. Both lipofectamine 2000 and lipofectamine rnaimax reagents are cationiclipid transfection reagents formulated for superior transfection efficiencies on a variety of cell types. The transfection efficacy of lipofectamine 2000 was compromised by. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0.
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. Lipofectamine rnaimax reagent thermo fisher scientific us. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from. Mammalian cells transfection protocoljavier calvo follow invitrogen lipofectamine 2000 protocol. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Invitrogen lipofectamine 3000 transfection reagent 0. The processing of dna for transfection and short interfering rna sirna. However, for cotransfection of sirna chimera and plasmid dna, lipofectamine 2000 should be used. The pretransfection almost completely abrogated the cellular. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Stealthsirna transfection protocol lipofectamine 2000.
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 chok1 cells with a reporter plasmid. Aug 07, 2012 add 2 microliters of lipofectamine ltx reagent to tube 1, 3 microliters to tube 2, 4 microliters to tube 3 and 5 microliters to tube 4. Cotransfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Mix gently and incubate for 20 min at room temperature to allow the sirna.
Lipofectamine 2000 is commonly used for sirna transfections. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338500 pdf. Transfecting sirna into mammalian cells using lipofectamine 2000. Optimized conditions for successful transfection of human. Advanced transfection with lipofectamine 2000 reagent.
Prewarm 50ml of optimum stored in cold room at 4c 10min. Highly efficient sirna delivery into primary cultures of rat pancreatic stellate cells. Baec, bovine aortic endothelial cells, sirna, lipofectamine rnaimax. 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. Nucleofection is a highly effective gene transfer technique for human. Combine the diluted sirna with the diluted lipofectamine 2000, mix gently, and incubate for 20 min at room temperature. We suppose that the antigenblocking capacity of scfv may play an. Abstract lipofectamine 2000 lipo2000 delivery system is commonly used for short interfering rna sirna transfection, whereas the cellular.
Interestingly, the rates of apoptosis induced by the s9rmediated sirna delivery or lipofectamine 2000 mediated sirna transfection did not differ in the presence of gefitinib. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Comparative nucleic acid transfection efficacy in primary. An efficient sirna transfection reagent in human embryonic stem cells, molecular biotechnology on deepdyve, the largest online rental service for. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle. Highest transfection efficiency in many cell types and formats e. 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. Im about to transfect viral rna from an in vitro transcription assay by cationic liposomes, into bhk cells and i was. Lipofectamine 2000 reagent thermo fisher scientific. Mix each tube well by vortexing or flicking the tube. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. I transfect my pc3 cell line with lipofectamine 2000 and my sirna plk1.
Mdamb231 cells were transfected with either control or ppp1r12asirna using lipofectamine 2000 invitrogen. I have used both lipofectamine 2000 and 3000 and always get the same results. Cotransfection of plasmid dna and sirnamirna is a technique popularly used by cell biologists, for applications such as knockdown rescue or. Preadipocytes were seeded in 6well plates, and sirna transfection was carried out 2 days after the confluence of preadipocytes. Effects of lipofectamine 2000sirna complexes on autophagy in. Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml. This reference provides general guidelines and procedures to transfect sirna. The microscopic images were manually analyzed to quantify. The intracellular trafficking mechanism of lipofectamine. Thee two most important factors in choosing transfecting reagent are. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Lipofectamine rnaimax transfection reagent delivery of sirna. Livecell imaging to compare the transfection and gene.
Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. 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. The best approach is to follow the guidance of the literature where possible and to test. Systematic screening of commonly used commercial transfection. I have used both lipofectamine 2000 and 3000 and always get the. Rnai in cultured mammalian cells using synthetic sirnas. Lipofectamine is formulated to give high transfection efficiency with little toxicity to cells. Highly efficient sirna delivery into primary cultures of rat. C2c12, mouse muscle myoblast, plasmid dna, lipofectamine 3000. No other sirna specific transfection reagent provides such easy and efficient sirna delivery in a. Culture vessel multiplication factor shared reagents dna transfection sirna transfection growth medium optimem medium for complexing dna p3000 reagent lipofectamine. Gccguggaggccuccuuca3 a scrambled 21nucleotide sirna with the sequence of 5. Protocol and application notes back to table of content. Add 6l of lipofectamine2000 from biostores and 100 l of.
Lipofectamine 2000 procedure for simultaneously transfecting. Photograph of expected results dna lipofectamine scaling up or down. Lipofectamine 2000sirna complexes cause endoplasmic reticulum. Cellspecific transfection protocols thermo fisher scientific us. Ntersirna nanoparticlemediated knockdown of gene expression xtremegene 360 transfection reagent if additional help is needed, please consult our technical services group at. Whats the difference between lipofectine and lipofectamine 2000 reagents. Qc specifications back to table of content lipofectamine 2000 is tested for the absence of microbial contamination using blood agar plates. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Add 2 microliters of lipofectamine ltx reagent to tube 1, 3 microliters to tube 2, 4 microliters to tube 3 and 5 microliters to tube 4. Transfection protocol of lipofectamine rnaimax can be. Lipofectamine rnaimax reagent is designed specifically for the delivery of sirna and mirna while lipofectamine 2000 reagent delivers dna or sirna with excellent. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1.
Cotransfection of plasmid dna and sirna mirna is a technique popularly used by cell biologists, for applications such as knockdown rescue or sirna mirna mediated knockdown of gene expression from a plasmid dna that is being delivered to the cell. They form liposomes taking up your dna or sirna positive charges of the liposome vs negative charge of nucleic acids. Lipofectamine 2000 transfection protocol 10cm plates reagents. Students ttest was used to compare the means of two groups and oneway.
Highly efficient sirna delivery into primary cultures of. Sep, 2005 invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. After the 5min incubation, combine the diluted sirna with the diluted lipofectamine 2000 total volume is 100. Transfection protocols are used in biological laboratories for introduction of foreign plasmid dna. Jul 24, 2007 transfecting sirna into mammalian cells using lipofectamine 2000. Whats the difference between lipofectine and lipofectamine. 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. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. There is a need to identify simple methods to genetically manipulate primary hepatocytes and. Actomyosin drives cancer cell nuclear dysmorphia and. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Lipofectamine 2000 and lipofectamine rnaimax transfection.
The following is a general protocol for use of dharmafect transfection. Lipofectamine 2000 transfection reagent from invitrogen. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. 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. Pool dishes of cells that have been prepared for transfection. I use 24well plates for transfections in triplicates. Jun 10, 2011 lipofectamine 2000 is commonly used for sirna transfections. Out of the five transfection systems tested, fugene and rnaimax.
The sirna knockdowns were performed using lipofectamine 2000 transfection reagent thermo fisher scientific, waltham, usa according to the manufacturers protocol. Transfection protocol lipofectamine 3000 transfection. Effects of lipofectamine 2000sirna complexes on autophagy. For this experiment, knockdown of endogenous luciferase was achieved in three engineered luciferase cell lines using lipofectamine. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Life technologies corporation andor its affiliates warrant their products as set forth in the life technologies general terms.
Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids. Dharmafect volumes per well represent guidelines and need to be optimized. Lipofectamine 2000 has been used successfully to transfect short interfering rnas sirna into. 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. Pkm2dependent glycolysis promotes the proliferation and. Human adipocytes stimulate invasion of breast cancer mcf7. The company has no samples for test, so, i would like to know if anyone can give a feedback on how they compare. Troubleshooting for transfection of the crisprcas9 using. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. The protocal is from thermo fisher scientific important guidelines for transfection 1. 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. Lipofectamine 2000 is tested for the absence of microbial contamination using blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. Lipofectamine 2000 has been used successfully to transfect short interfering rnas sirna into mammalian cells for rna interference rnai studies gitlin et al.
Lipofectamine 2000 transfection reagent dartmouth college. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. How does transfection with lipofectamine 2000 work. Lipofectamine 2000 is used to deliver the sirnas into hek293 cells. However, few studies have examined cellular responses to this delivery system. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Rubi fructus rubus coreanus inhibits differentiation to. Dna complex stability nov052012 i have already prepared the complex dna. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Photograph of expected results dnalipofectamine scaling up or down. Invitrogen lipofectamine 2000 transfection reagent. Invitrogen lipofectamine 3000 transfection reagent lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results.