I use 24well plates for transfections in triplicates. Rapid and highly efficient mammalian cell engineering via. An efficient sirna transfection reagent in human embryonic stem cells, molecular biotechnology on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Rapid and highly efficient mammalian cell engineering via cas9 protein transfection. A volume of 2 ul of lipofectamine 2000 do you think will be enough for an overnight transfection in dmemno pens strep 100 nm sirna. I transfect my pc3 cell line with lipofectamine 2000 and my sirna plk1 and with the controls. When i transfect hela cells with lipofectamine 2000, cell size of 1015%.
There is a need to identify simple methods to genetically manipulate primary hepatocytes and. Mix gently and incubate for 20 min at room temperature to allow the sirna. Lipofectamine 2000 transfection protocol 10cm plates reagents. Prewarm 50ml of optimum stored in cold room at 4c 10min. After the 5min incubation, combine the diluted sirna with the diluted lipofectamine 2000 total volume is 100. I have predesigned sirna to my gene of interest from dharmacon.
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. Dharmafect volumes per well represent guidelines and need to be optimized. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Has anyone tried sirna transfection with lipofectamine3000. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. 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 hela, swis 3t3, 293t with lipofectamine 2000 1. Graphpad prism software and students ttest was used to. Highly efficient sirna delivery into primary cultures of. Make a test array and show which concentration has no effect on your worms. The ability to alter microrna mirna abundance is crucial for studying mirna function. Lipofectamine rnaimax reagent thermo fisher scientific us. C2c12, mouse muscle myoblast, plasmid dna, lipofectamine 3000.
Creating an sirna is a simple matter of annealing two chemically synthesized oligoribonucleotides to generate a molecule with 19 paired bases and two overhanging nucleotides on the 3. Although this amount is good for a wide range of sirna concentrations, if more sirna is transfected, the amount should be increased accordingly. I ordered now some reagents from lipocalyx viromer and will test hose. The protocal is from thermo fisher scientific important guidelines for transfection 1. The processing of dna for transfection and short interfering rna sirna. Highly efficient sirna delivery into primary cultures of rat pancreatic stellate cells. Cells were transfected with sirna and lipofectamine2000 according to the manufacturers instructions. Okita in the laboratory of professor yamanaka at the center for ips cell research and application cira, kyoto university. This reference provides general guidelines and procedures to transfect sirna into mammalian cells using lipofectamine 2000.
However, few studies have examined cellular responses to this delivery system. Apr 15, 2014 the use of dharmafect 2 and dharmafect 4 resulted in sirna uptake by a high proportion of bmdm, but caused considerable cell toxicity. Systematic screening of commonly used commercial transfection. Optimemin ul, equal for dna and lipofectamine lipofectaminein ul 25. Briefly, cells were seeded in a 24wellplate at a density of 0. Lipofectamine 3000 reagent can deliver dna into dif. Invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Suggested transfection conditions are provided for a number of mammalian cell lines as a starting point. Whats the difference between lipofectine and lipofectamine. Livecell imaging to compare the transfection and gene.
For this experiment, knockdown of endogenous luciferase was achieved in three engineered luciferase cell lines using lipofectamine. Jun 10, 2011 lipofectamine 2000 is commonly used for sirna transfections. Lipofectamine rnaimax transfection reagent delivery of sirna. On measuring mirnas after transient transfection of mimics. Im about to transfect viral rna from an in vitro transcription assay by cationic liposomes, into bhk cells and i was. Lipofectamine rnaimax reagent is designed specifically for the delivery of sirna and mirna while lipofectamine 2000 reagent delivers dna or sirna with excellent. Lipofectamine 2000 reagent thermo fisher scientific. Sep, 2005 invitrogens lipofectamine 2000 is one of many cationic lipidbased transfection reagents presently on the market. Hiperfect, interferin, lipofectamine 2000, lipofectamine rnaimax, xtremegene and dharmafect 3 promoted good uptake of sirna by bmdm, without causing significant cell death. When i transfected sirna with lipofectamine with the amounts that appear in the protocol, all my cells died. In the absence of sirna, lipofectamine 2000 induced differential expression of over 500. Effects of lipofectamine 2000sirna complexes on autophagy. Invitrogen lipofectamine 2000 transfection reagent. Lipofectamine 2000 is commonly used for sirna transfections.
The intracellular trafficking mechanism of lipofectamine. Combine the diluted sirna with the diluted lipofectamine 2000, mix gently, and incubate for 20 min at room temperature. If using reagents suitable for the transfection of dna and rna. The intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the cotransfection of sirna and plasmid dna. Stealthsirna transfection protocol lipofectamine 2000. Start any new transfection by testing the recommended four concentrations of lipofectamine 2000 reagent to determine an optimum amount. We have also used lipofectamine 2000 to introduce sirna into primary synoviocytes and obtained a comparable transfection. Jul 24, 2007 this reference provides general guidelines and procedures to transfect sirna into mammalian cells using lipofectamine 2000. Hiperfect transfection reagent qiagen for sirna transfection in adherent cells.
Troubleshooting for transfection of the crisprcas9 using. Advanced transfection with lipofectamine 2000 reagent. Given these uncertainties, the most common synthetic rnai trigger in mammalian cells is the sirna. For using lipofectamine 2000 reagent, the above plasmids were mixed with 30 ul. 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. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. The following is a general protocol for use of dharmafect transfection. Comparison of small interfering rna sirna delivery into. Effects of lipofectamine 2000sirna complexes on autophagy in. 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. An improved method for increasing the efficiency of gene. Rnai in cultured mammalian cells using synthetic sirnas. Pool dishes of cells that have been prepared for transfection. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids.
Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. Optimemin ul, equal for dna and lipofectamine lipofectamine in ul 25. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Hi all, i am doing sirna knockdown for the first time tomorrow. Highly efficient sirna delivery into primary cultures of rat. Lipofectamine 3000 reagent for pluripotent stem cell applications.
Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. At the foundation of the popularity of this broadspectrum reagent is the reliably high efficiency and simple protocol for success across a broad range of cell lines figure 1. The best approach is to follow the guidance of the literature where possible and to test the panoply of available transfection methods where necessary. Thee two most important factors in choosing transfecting reagent are. Start any new transfection by testing the recommended. On measuring mirnas after transient transfection of mimics or. Students ttest was used to compare the means of two groups and oneway. Lipofectamine 2000 transfection reagent from invitrogen. I got recommendations from many to use lipofectamine 2000 transfecting reagent from invitrogen rather than the ones provided by dharmacon. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Using cx43 shrna lentivirus combined with lipofectamine 3000 transfection. 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. Lipofectamine plasmid transfection bridges lab protocols.
Both lipofectamine 2000 and lipofectamine rnaimax reagents are cationiclipid transfection reagents formulated for superior transfection efficiencies on a variety of cell types. Transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. I usually use 56 ulwell lipofectamine 2000 for an sirna concentration of 150 nm in a 24well plate as suggested by the vendor. The success of these manipulations is often assessed using qpcr, but this does not accurately report the level of. Whats the difference between lipofectine and lipofectamine 2000 reagents. Lipofectamine 2000 dna transfection reagent protocol. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. 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. However, sirna delivery into primary cells, especially primary macrophages, is often considered challenging. The transfection efficacy of lipofectamine 2000 was compromised by its high. I have used both lipofectamine 2000 and 3000 and always get the same results. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. The microscopic images were manually analyzed to quantify.
Photograph of expected results dnalipofectamine scaling up or down. Qc specifications back to table of content 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. Instead the vast majority of mirna transfected with either hiperfect, rnaimax or lipofectamine 2000 data. Lipofectamine 2000 procedure for simultaneously transfecting.
The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy. Photograph of expected results dna lipofectamine scaling up or down. On measuring mirnas after transient transfection of mimics or antisense inhibitors. However, for cotransfection of sirna chimera and plasmid dna, lipofectamine 2000 should be used.
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. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Comparative nucleic acid transfection efficacy in primary. Add 6l of lipofectamine2000 from biostores and 100 l of.
For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Lipofectamine 2000 and lipofectamine rnaimax transfection. The comparative utility of viromer red and lipofectamine. 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. Its very common to transfect sirna with lipofectamine3000. 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. There is a need to identify simple methods to genetically manipulate primary hepatocytes. A scrambled sirna that does not target any gene was used as the negative control. The example which will be illustrated is transfection of rat pscs with hsp90 specific sirna using lipofectamine 2000. Nucleofection is a highly effective gene transfer technique for human. Cho k1, hamster ovary, plasmid dna, lipofectamine ltx, 15338100 15338500 pdf. This shows that the lipofectamine 2000 concentration should be optimized for each cell type. Add the oligomerlipofectamine 2000 complexes from step 2. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping.
Optimized conditions for successful transfection of human. No other sirna specific transfection reagent provides such easy and efficient sirna delivery in a. 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. Protocol 6 well dish plate cells the day before so that they are at 9095% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. May 11, 2016 the intracellular trafficking mechanism of lipofectaminebased transfection reagents and its implication for gene delivery. Baec, bovine aortic endothelial cells, sirna, lipofectamine rnaimax.
Cellspecific transfection protocols thermo fisher scientific us. Life technologies corporation andor its affiliates warrant their products as set forth in the life technologies general terms. Lipofectamine 3000 reagent thermo fisher scientific. If you are using these cell lines or another mammalian cell line, we recommend optimizing transfection conditions to. 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.
664 1555 492 148 678 927 786 380 54 769 1087 1512 243 85 459 742 159 1059 1032 4 592 802 212 904 1236 425 208 795 301 81 338 1074 1228 605 1548 486 1056 331 1368 1188 993 1329 131 853 435 1096 577 1224 436 1249