Forrest cardiff university a thesis submitted for the degree of doctor of philosophy 20 supervisors. This makes lipofectamine rnaimax reagent easy to optimize for the lowest sirna concentration while reducing cytotoxicity in your experimental system. L of medium containing 20% fbs with the wellmate dispenser. Vana mimics and inhibitorsnovel tools for mirna functional analysis. L of serumfree media using a wellmate dispenser thermo scienti. Lipofectamine 2000 procedure for simultaneously transfecting. Plate cells so they will be 6080% confluent at the time of transfection. However, delivery of the cas9 rnps by neon electroporation was effective across all different cell lines tested and often was the only method that could produce indels.
Transfection of mcf7 cells with lipofectamine 2000 and isolation of genomic dna abstract. Lipofectamine transient transfection of cos7 cells to analyze. Lipofectamine rnaimax transfection reagents introduction life technologies is committed to providing researchers and scientists the best products, enabling cutting edge technology while keeping the environment in mind. Plasmid transfection and expression in p388 cells angelo mancino, birgitta r. From our experiences, we noticed that lipofectamine rnaimax is superior to lipofectamine 2000 to the extent of cytotoxicity and rnai efficiency, and that cytotoxicity of lipofectamine 2000 varies lot by lot. Lipofectamine transient transfection of cos7 cells to. This unpublished thesisdissertation is of the author andor third. Dear all, i am very new in the field of cell biology and i experience a lot of problems.
Prepare 6 well plates by adding bakedsterile 22x22 1. Rna interference methodology suppresses specific gene expression, thus mimicking lossoffunction mutation and enabling in vitro and in vivo gene function analysis. Mix and incubate for 20 minutes at room temperature. Cells cultured in 6well plates were transfected at 3050% confluence using lipofectamine. Lipofectamine rnaimax, a new transfection reagent, has been confirmed high efficiency in delivering small interfering rna sirna into mesenchymal stem cells and neural stem cells. Lipofectamine rnaimax rnaimax, a new cationic lipofection reagent, has shown higher efficiency in delivering mirna into many stem cells and cell lines zhao et al. Microrna203 inhibits proliferation and induces apoptosis of. Lipofectamine rnaimax reagent enables maximal knockdown and excellent cell viability across a 10fold concentration range of the reagent.
Lipofectamine rnaimax reagent thermo fisher scientific assets. Lipofectamine rnaimax, what medium for transfection. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types. There are several ways in which to introduce cas9guide rna complexes into cells. Thee two most important factors in choosing transfecting reagent are.
Highest transfection efficiency in many cell types and formats e. It takes nearly 120 barrels of crude oil equivalents and 150 mwh of power annually to make the 9,000 kg of eps coolers needed to ship these reagents1,2. The lipofectamine rnaimax reagent is designed specifically for the delivery of sirna and mirna, and lipofectamine messengermax reagent delivers mrna. Product is guaranteed for 6 months from the date of shipment if stored properly. Feb 18, 2016 lipofectamine crisprmax is more robust than lipofectamine rnaimax and several other transfection reagents in delivery of cas9 proteingrna complexes into a variety of cell lines.
Add the rnai duplexlipofectamine rnaimax complexes to each well containing cells. Transfection of cas9 rnp ribonucleoprotein into adherent. Lipofectamine rnaimax reagent thermo fisher scientific. Click add files and select the files you want to include in your pdf. Incubate the cells 2448 hours at 37c in a co2 incubator until you are ready to assay for gene knockdown. The crisprcas system is a genome editing technology that works with a cells endogenous repair mechanism. I am thinking about the cell density or the lipofectamine rnaimax amount that i should use. How much lipofectamine rnaimax shoiuld be used for sirna. The transfection is designed for one rna amount combined with one amount of lipofectamine rnaimax. Improved delivery of cas9 proteingrna complexes using. This thesis is submitted by marie ann voice to the university of birmingham for the degree of. Lipofectamine 2000 and 3000 summary in this comparative study, transfections were performed with dnain and compared to the current market leader, lipofectamine 2000 lf2k, using a series of cultured celllines and primary cells. Transfecting sirna using lipofectamine rnaimax pin.
Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. The protocal is from thermo fisher scientific important guidelines for transfection 1. Lipofectamine 2000 transfection reagent dartmouth college. I want to transfect mammalian cells with a large gene 8 kb fused with gfp. Lipofectaminea rnaimax reagent thermo fisher scientific. Lipofectamine rnaimax reagent thermo fisher scientific us.
Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Formulation of lipofectamine 2000 and lipofectin signagen blog. Lipofectamine rnaimax troubleshooting ive been having trouble getting efficient sirna knockdown of my protein of interest using lipofectamine rnaimax. The prepared mix is enough to have triplicates 96well, duplicates 24well, and single well 6well transfections, and account for pipetting variations. Andersen university of aarhus, department of molecular biology c. Lipofectamine 2000 reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells. Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects.
Lipofectamine 2000 and lipofectamine rnaimax transfection. For the hardtotransfectcell line such as hepg2, we recommend the reverse transfection. Add 2 mls of media to each well and seed with 400,000 cos7 cells per coverslip. Pool dishes of cells that have been prepared for transfection. The following cell lines have been successfully transfected with the indicated transfection reagents.
Make sure and evenly distribute the cells on the coverslips by swirling plate. I would like to use lipofectamine rnaimax for transfection of cows luteal cell with sirna duplex in 24 well plates. Materials and methods page 52 figure mf1 determination of quality and quantity of genomic dna from cervical precancer and cancer and normal control tissues. For additional information on scaling your transfection reaction, see page 4. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas e. Prepare a mixture of the transfection plasmids with optimem 250ulwell. Plasmid measure concentration 293 t packaging cells at 1. How to merge pdfs and combine pdf files adobe acrobat dc. The transfection reagent and sirna were complexed for 45 min at ambient temperature before the addition of 1,500 cells in 20. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Refer to the cell lines database for a list of cell types successfully transfected. Follow manufacturers handbook protocol unless otherwise indicated. The main components of the system are cas9, which is an endonuclease, and a guide rna or grna. Lipofectamine reagents go online to view related products.
We would like to show you a description here but the site wont allow us. Mix lipofectamine rnaimax gently before use, then dilute 3. Lipofectamine 2000 transfection reagent from invitrogen. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. This gives a final volume of 600 l and a final rna concentration of 10 nm.
Finding out adapters information in small rna seq data. Transfection of cas9 rnp ribonucleoprotein into adherent cells using the lipofectamine rnaimax overview. Culture vessel multiplication factor1 shared reagents dna transfection sirna transfection vol. Lipofectamine 2000 and lipofectamine 3000 reagents deliver dna or cotransfect with excellent transfection performance for protein expression, gene silencing, and functional assays. Lipofectamine 2000 reagent thermo fisher scientific. 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. Combine the diluted sirna duplex with the diluted 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. Typical rnaimax transfection procedure transfect cells according to the following table. Scaling up or down lipofectamine 3000 reagent transfections. Lipofectamine rnaimax transfection reagent gives maximal knockdown and excellent cell viability across a 10fold concentration range of the reagent figure 2.
Transfection using effectene, lipofectamine, and polyfect reagents many reagents are now available for optimization of cell transfection efficiency. Hek293 cells or other cell line at 3070% confluency. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. By shipping these reagents at ambient temperatures. Comparison of transfection through lipofectamine and polyeth. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Hepatocellular carcinoma sixth most common cancer third leading cause of cancer related deaths early stage treatments liver transplantation and surgical resection metastasis and recurrence dna will be transfected into the huh7 cell line ability to grow without serum growth.
An external file that holds a picture, illustration, etc. I use 24well plates for transfections in triplicates. Zhao m1, yang h, jiang x, zhou w, zhu b, zeng y, yao k, ren c. Lipofectamine crisprmax is more robust than lipofectamine rnaimax and several other transfection reagents in delivery of cas9 proteingrna complexes into a variety of cell lines. Cas9 nuclease may be used in vivo to create targeted genome modifications. Antisense oligonucleotides bind and inhibit mirnas. Genomic dna isolated from cervical normal, precancer and cancer tumor tissue biopsies was. Xinxin liu, updated august 2014 prsv 10 ug mix measure concentration pmdl 10 ug vsv 10 ug plenti. Invitrogen lipofectamine rnaimax transfection reagent. For micrornaseq data the fastqc pipeline works quite fine, but there are two notes, i would like to add. The following is a general protocol for use of dharmafect transfection reagents to deliver sirna into cultured mammalian cells. Dmriec reagent, lipofectamine 2000, optimem i reduced serummedium. I have successfully done this knockdown many times in the past, but i havent been able to get it to work recently.
Lipofectamine rnaimax reagent thermo fisher scientific uk. Lipofectamine 2000, 3000, messengermax, and rnaimax. Click, drag, and drop to reorder files or press delete to remove any content you dont want. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. For the cell lines easy to transfection, you can use either of the procedures reverse or forward transfection. This makes lipofectamine rnaimax reagent easy to optimize for the lowest sirna concentration that can be used while reducing cytotoxicity in your experimental system.
You can merge pdfs or a mix of pdf documents and other files. Invitrogen lipofectamine 3000 transfection reagent 0. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. Hello there, i purchased 5 sirna sequences from sigma and tried to transfect hek cells using lipofectamine. Comparison between lipofectamine rnaimax and genmute.
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