Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Advanced transfection with lipofectamine 2000 reagent. Jayarajan, charite berlin, center for cardiovascular research germany % gfp positive cells complete medium serum and antibioticfree medium 14. Highest transfection efficiency in many cell types and formats e. The vectorrelated influences of autophagic microrna. Exceptional broad spectrum transfection cutting edge delivery of plasmid dna andor small rnas sirna, mirna, and crispr guide rna outperforms lipofectamine 2000 in 28 of 41 tested cell lines.
Do you have experience using an efficient transfection reagent for. I use 24well plates for transfections in triplicates. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. Cells were transfected with a nonlabelled and an alexa fluor 488 green labelled, nonspecific sirna using lipofectamine 2000 lf2000, screenfect a. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. M phosphate buffered saline pbs plasmid dna high quality sirna 20. In biomedical applications, this process of rna interference has encouraged researchers to study ways in which this process can be utilized to shut down or effectively incapacitate a defective or nonwanted genes ability to replicate. Unlike for conventional dna transfection, transport of the nucleic acid into the cell nucleus is not necessary for rna transfection. Comparative nucleic acid transfection efficacy in primary. Reverse transfection protocol for sirna in 96well plates using transittko transfection reagent before you start. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Hela, a549, panc1, mb231 total transfection volume. Lipofectamine 2000 reagent thermo fisher scientific. I am trying to knockdown cmyc andor ras using mirna, let7.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Lipofectamine 2000 and lipofectamine 3000 reagents deliver dna or cotransfect with excellent transfection. On measuring mirnas after transient transfection of mimics or. Add 6l of lipofectamine2000 from biostores and 100 l of. Luciferase expression was compared at 24 hours posttransfection using a standard luciferase assay. Synthetic mirna mimics from dharmacon or shanghai genepharma were transfected into cells following the dharmafect1 transfection protocol t2001. When i transfected sirna with lipofectamine with the amounts that appear in the protocol, all my cells died. Lipofectamine 2000 is primarily used for delivery of plasmid dna, but it also works well for delivery of sirna and mirna mimics and of course co. 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. Synthetic mirna mimics from dharmacon or shanghai genepharma were transfected into cells following the dharmafect1 transfection protocol t200103. A comparison showing transfection efficiency of polyjet reagent vs. Transfection in multiwell plates transfection qiagen. Lipofectamine 2000 lipo was obtained from invitrogen corp. Microscopic observations of rat primary astrocytes transfected with pmaxgfp using.
Establishment of lipofection for studying mirna function in. Lipofectamine 2000, and lipofectamine 2000 cd x broad spectrum dna deliveryachieve high expression in many cell types, including hardtotransfect cells and insect cells x animal origin freetransit2020 provides high performance with maximum compatibility description transit2020 reagent is a new and versatile transfection solution for. Adipocytes, however, tend to be resistant to transfection and there is often a need to resort to viral transduction or electroporation. In order to study mirna function in human adipocytes, we aimed for the modulation of mature mirna concentration in these cells. Rnai transfection protocol see page 2 to view guidelines for transfecting mirnas using lipofectamine rnaimax reagent. Lipofectamine 2000 transfection reagent, invitrogen. Prewarm 50ml of optimum stored in cold room at 4c 10min. High efficiency transfection reagent at a reasonable price.
Transfection of microrna mimics should be used with caution. Transfection the delivery of dna or rna into eukaryotic cells is a powerful tool used to study and control gene expression. Viromer blue 50 pmol lipofectamine 2000 100 pmol vb01lb00, vb01lb01 primary astrocytes. 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. Feb 06, 2010 i am trying to knockdown cmyc andor ras using mirna, let7. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Ive been working with sirnas just for a couple of weeks and i have more doubts that answers. We are using lipofectamine for transfecting synthetic mirna mimics for the mirna. Lipofectamine 2000 and lipofectamine rnaimax transfection. Quantitative pcr following mirna mimic transfection or addition post lysis. Three cell densities of hepg2 cells were transfected with gapd sirna 100 nm using a range of volumes 0. Prior to transfection, medium was replaced with fresh growth medium.
Pdf establishment of lipofection for studying mirna. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Biomodule transfection and control user manual unit with. Lipofectamine rnaimax has a broad peak of activity. Plgabased dual targeted nanoparticles enhance mirna. Do not add antibiotics to media during transfection as this causes cell death. Lipofectamine 2000 right panel around 5% efficiency.
Add the oligomerlipofectamine 2000 complexes to each well containing cells and medium. L reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. Transfection the delivery of dna or rna into eukaryotic cells is a. Lipofectamine 2000 transfection reagent dartmouth college. Mix gently and incubate for 20 minutes at room temperature solution may appear cloudy. The vectorrelated influences of autophagic microrna delivery. Instead the vast majority of mirna transfected with either hiperfect, fig. Lipofectamine rnaimax reagent thermo fisher scientific. Media were replaced with antibioticsfree media 612 h before transfection. With proprietary shaved cell transfection protocol, polyjet left panel gives up to 70% gfp positive cells vs. Perform reverse transfection by preparing complexes inside the wells, and then adding cells and medium. Therefore, the high level of transfected mirna detected by qpcr is largely attributable to their.
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. In this video, we will perform an sirna transfection experiment using. Can i use the lipefectamine2000 for mimic mirna transfection. Sgbs preadipocytes were seeded in a 12well plate at a density of 7900 cellscm 2 and transfected the following day with final concentrations of 20 nm simirna and 0. Guangzhou, china, while a mirna mimic with random sequences nc mir was served as the negative controls. Transfection amounts per well use 10 nm mirna duplex as a starting point.
Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Transfection and genome engineering fisher scientific. The intracellular trafficking mechanism of lipofectamine. Lipofectamine 2000, 3000, messengermax, and rnaimax reagents are cationiclipid transfection reagents formulated for superior transfection efficiencies on a variety of cell types. Lipofectamine 2000, 3000, messengermax, and rnaimax. Lipofectamine 2000 is primarily used for delivery of plasmid dna, but it also works well for delivery of sirna and mirna mimics and of course codelivery. In recent years it has become clear that microrna mirna are crucial. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. 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. Dec 02, 2015 transient transfection of mirna mimics. 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. For additional information on scaling your transfection reaction, see page 4.
I use lipofectamine 2000 as my standard transfection reagent of choice and. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. However, transfection using lipofectamine 2000 resulted in lower. Request pdf advanced transfection with lipofectamine 2000 reagent. Invitrogen lipofectamine 2000 transfection reagent. Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Max reagent from life technologies works awesome just follow their manual. Despite this, a satisfactory mechanismbased explanation of their. Aug 07, 2012 how to perform sirna transfection with lipofectamine rnaimax protocol. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Primary neurons, sirna, and highthroughput applications lipofectamine 2000 is a. The transitx2 dynamic delivery system mirus bio and lipofectamine 2000 transfection reagent thermo fisher scientific were used to transfect plasmid dna encoding luciferase into 41 different cell types at three reagenttodna ratios.
Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Once reconstituted in nucleasefree water, the mirna is ready to transfect and can be used at your choice of final concentration. Stealthsirna transfection protocol lipofectamine 2000. Lipofectamine tm 2000 lip2000 transfection reagent was used as a positive control.
Jan 24, 20 instead the vast majority of mirna transfected with either hiperfect, fig. Highthroughput microrna mirna transfection, where 319 premirs were transfected, was applied in the study of estrogen receptor signaling in breast cancer cell lines leivonen et. A sample protocol is listed here for transfection experiments performed in 6well plates. Adipocytes, however, tend to be resistant to transfection and there is often a need to resort to viral transduction or. Establishment of lipofection for studying mirna function. Optimizing transfection of mirna sirna, microrna and rnai. Both lipofectamine 2000 and lipofectamine rnaimax reagents are cationiclipid transfection reagents formulated for superior transfection efficiencies on a variety of cell types. Hek293ft cells were transfected with gfp vector pegfpn3 by polyjet left panel and lipofectamine 2000 right. Cells were transfected with a nonlabelled and an alexa fluor 488 green labelled, nonspecific sirna using lipofectamine 2000 lf2000, screenfect a sfa. A mirna mimic mir494 based on sanger mirbase was synthesized by guangzhou ribobio co. 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. Genscript recommends using lipofectamine 2000 for all transfections. So i asked one of my lab mates and he told me to use just 1ul. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used.
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