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