FAQ
DNA in vitro transfection: PROTOCOLMy cells are fragile and do not grow well in absence of serum. May I try transfection with serum?
In contrast to many other transfection reagents, the efficiency of both jetPRIME™ and jetPEI™ is not affected by the presence of serum nor by the presence of antibiotics. The complexes can therefore be added directly to the complete medium.
Can I use antibiotics during transfection with jetPRIME™?
The presence of antibiotics does not reduce jetPRIME™ efficiency for the transfection of plasmid DNA in many various cell lines. The routine validation protocol for each new batch of jetPRIME™ is always performed in complete medium (i.e in the presence of serum and antibiotics).
Transfection efficiencies can usually be improved by using smaller volume of culture medium. Another method consists in gentle centrifugation of the culture plate (5 min at 280g). Alternatively, higher amounts of DNA and/or higher amount of reagent can also increase transfection efficiency.
For optimal transfection with jetPRIME™, we recommend transfecting cells at 60% to 80% confluency.
For optimal transfection with jetPEI™, we recommend using the cells at 50% to 60% confluency. Please refer to Table 3 in the jetPEI™ transfection protocol for the recommended number of cells to seed according to the cell culture plate.
In general, the amount of DNA should be proportional to the total number of cells. Refer to the regular jetPEI™ protocol indicating the amount of DNA and jetPEI™ needed according to the size of culture dish used (Table 3).
The total amount of DNA should remain the same as when transfecting only one plasmid (e.g. 1 µg/well in a 24-well plate). You simply need to use less of each plasmid. For example 0.5 µg of each plasmid when transfecting two. Note however that various plasmids may not be required in equivalent amounts (i.e. a plasmid encoding for the protein of interest versus a plasmid encoding for a transcription factor etc…). The amount of each plasmid should not be less than 10% of the total DNA amount transfected per well. Thus it is recommended to determine and optimise the amount of DNA required for each plasmid according to their expression level.
Can jetPEI™ be used for transient as well as stable transfections?
Yes, jetPEI™ can be used for stable as well as transient transfections. Start selection with the appropriate antibiotic 24 h to 48 h after transfection. In order to determine the correct amount of antibiotic for clone selection, we recommend performing a killing curve with your cell line using your culture conditions. We also recommend linearizing the expression plasmid prior to transfection in order to facilitate its integration into the genome.
What would you suggest to reduce the cytotoxicity of complexes jetPEI™/DNA on fragile cells (e.g. primary fibroblasts)?
In order to reduce the cytotoxicity caused by transfections, we suggest the following:
- change media 4 h post-transfection to remove jetPEI™/DNA complexes
- decrease the quantity of DNA and jetPEI™ used per well, keeping the same N/P ratio (e.g. in a 24 well-plate use 0.5 µg of DNA and 1 µl of jetPEI™ per well instead of 1 µg of DNA and 2 µl of jetPEI™, keeping the N/P=5)
- increase the percentage of serum in the media used during transfection (e.g. 15% instead of 10%).
Can I store my jetPEI™/DNA complexes for 24 h at 4°C?
jetPEI™/DNA complexes prepared in NaCl are only stable for 2 h at room temperature. However, complexes prepared in 5% glucose solution, such as for in vivo-jetPEI™, can be kept up to 24 h at 4°C.
Is there a plasmid size restriction for DNA transfection using jetPEI™?
There is no size restriction for DNA transfection using jetPEI™. jetPEI™ is a polyamine compound which is able to compact and deliver small and large DNA molecules. The largest reported plasmid transfected in human cells using PEI is a Yeast Artificial Chromosome (YAC) of 2.3 Mb intact into human cells ( P. Marschall, N. Malik and Z. (1999) Larin Gene Therapy 6, 1634-1637). However, keep in mind that for the same amount of DNA, the gene copy number will be less for large DNA such as YACs than for smaller plasmids.
How long does jetPEI™-FluoF or jetPEI™-FluoR fluoresce in cells?
The fluorochromes (F or R) conjugated to jetPEI™ are always fluorescent. During the process of transfection, the fluorescence will be visible as long as jetPEI™ is present in the cells. However, note that the fluorescence is degraded by the laser of the microscope and by natural light. Thus leaving transfected cells exposed to the microscope light for too long drastically decreases fluorescence intensity. As a result, the reagents should be stored in the dark. In experiment using jetPEI™-Fluos to transfect plasmid DNA, we observed fluorescence up to 72 hours after transfection.
How is the plasmid delivered from the cytoplasm to the nucleus when transfection is performed with jetPEI™?
The mechanism of plasmid delivery from the cytoplasm to the nucleus with jetPEI™ is mainly a passive one occurring during cell division.
Has jetPEI™ been already tested for transfection into vegetal cells?
jetPEI™ has been tested for the transfection of plant cells. Vegetal cells have a cellulose rich-membrane that prevents jetPEI™/DNA complexes entering the cells. Even at the protoplasts stage where the cellulose envelope has been removed, complexes were not able to penetrate into the cytoplasm.
What would you recommend using to transfect plasmid DNA into HUVEC cells?
For HUVEC cells, we would recommend using jetPEI™-HUVEC, a reagent specifically designed for this purpose. Together with the product you will receive a detailed protocol indicating HUVEC culture conditions and transfection procedure in order to obtain a 50% transfection efficiency.
How efficient is jetPEI™ for the transfection of Jurkat cells?
jetPEI™ is among the most efficient chemically based transfection reagents for the transfection of lymphocytic cell lines such as Jurkat, even if less efficient than electroporation techniques, especially for primary lymphocytes. Transfection of Jurkats with jetPEI™ has been previously reported (Goffin V., et.al., 2005, Nucleic Acids Res 33: 4285-4310. Soller M., et al., 2006, J Leukoc Biol 79: 235-243, Abbady A. et al., 2005. Biochem Pharmacol 70:570-579.). Optimal protocol conditions are also available here.
Can I use jetPEI™ on primary neurons culture?
Neurons are very difficult to transfect mainly because they do not divide in culture. However, jetPEI™ and similar PEI have been used for the transfection of neurons in vitro as well as in vivo :
- Boussif O et al. (1995), Proc Natl Acad Sci U S A 92: 7297-301
- Lambert RC et al. (1996), Mol Cell Neurosci 7: 239-246
- Kung AY et al. (2001), Neurosci Lett 309: 202-206
- Horbinski C et al. (2001), BMC Neurosci 2, 2
- Lemkine GF et al. (2002), Mol Cell Neurosci. 19:165-74.
Our team has developed has developed a specific protocol for DNA transfection of primary neurons. Please contact our technical support for more details.
What is the transfection efficiency of jetPEI™-macrophage in monocyte-derived human macrophages?
The transfection efficiency of jetPEI™-macrophage on human macrophage is approximately 30% according to the jetPEI™-Macrophage protocol or 107 RLU/mg protein. It is essential to grow the human primary macrophage derived from blood monocytes for 7 to 10 days with 100 U/ml of GM-CSF prior to transfection in order for the cells to express functional mannose receptors (C. Furman et al., (2004) Free Radic Biol Med 37: 71-85).
What type of compound is jetPEI™?
jetPEI™ is composed of a water-soluble polyamine. In contrast to the other class of transfection reagents, i.e., cationic lipids, it does not aggregate upon long-time storage, hence allowing accurate reproducibility. It also forms stable complexes with DNA, facilitating handling and reproducibility. Finally, due to the proton sponge mechanism used to release nucleic acid into the cytoplasm, it is very efficient.
What is the molecular weight of the linear PEI contained in jetPEI™?
jetPEI™ is composed of a linear polyethylenimine (PEI) whose molecular weight is proprietary information. Extensive optimization experiments have been performed in order to select the linear PEI giving the optimal DNA delivery with the most reduced toxicity in vitro and in vivo.
What is the difference between jetPEI™ and in vivo-jetPEI™?
jetPEI™ (for in vitro use) and in vivo-jetPEI™ are based on the same active molecule, however they have been specifically developed for each application. Thus they differ in formulation, concentration, purity and validation tests (in vivo-jetPEI™ has been tested for absence of endotoxin and validated for in vivo gene delivery in mice).
What is the composition of jetPEI™?
jetPEI™ is a completely synthetic product synthesised by our chemists, totally free of material of animal origin. It does not contain any other component than the polymer and water.
Which type of cell lines has INTERFERin™ already been successfully tested on?
INTERFERin™ has been successfully used for various adherent and non-adherent cell lines such as Hela, A549, BNL-CL2, 293, BHK-21, CHO and K562. INTERFERin™ has also been used on several primary cells such as primary human fibroblast and primary human hepatocytes. For more details, please refer to the Cell Transfection Database.
What is INTERFERin™?
INTERFERin™ is a new generation of non liposomal cationic amphiphile transfection reagent. It was developed for the delivery of siRNA into mammalian cells in culture. INTERFERin™ provides more than 90% silencing efficiency at 1 nM siRNA.
What would you recommend for siRNA delivery in vivo?
We do offer solutions for in vivo siRNA delivery. These depend on the animal model used, the route of injection and the targeted organ. Please contact our technical support who will provide you with a solution that best suits your requirements.
Would you recommend jetPEI™-Macrophage or INTERFERin™ for transfecting human monocytes, macrophages and dendritic cells with siRNA in vitro?
For in vitro siRNA transfection we always recommend using INTERFERin™. We have no comparative data with jetPEI™ as it is not designed for in vitro siRNA transfection. Please contact our technical support who will glad to provide you with an optimized protocol for your application
What is PULSin™ reagent?
PULSin™ contains a cationic amphiphile molecule capable of forming complexes with many proteins, including antibodies or peptides, resulting in a positively charged coat. These PULSin™/proteins complexes bind to syndecans, a molecule presents in virtually all cells membrane, allowing entry in the cell and subsequent escape of the protein from the endosome to the cytoplasm.
Which cells have been tested using PULSin™?
Established cell lines from various types such as HeLa and NIH-3T3, suspension cells such as Jurkat and primary cells such as human keratinocytes have all been successfully tested for the delivery of r-phycoerythrin, the positive control protein included in the kit (for an exhausitve list please refer to Cell Transfection Database.
Which proteins have been tested using PULSin™?
Negatively charged protein at physiological pH (protein with pI ranging from 0 to 7.4) with size ranging from 40 to 250 KDa have been successfully delivered using PULSin™. Moreover, various
antibodies recognizing different cell components such as anti-giantin, anti-vimentin and anti-tubulin have also been successfully delivered still recognising their target. Peptides have also been successfully delivered (for details please refer to PULSin™ page)
With PULSin™, is the antibody or protein only delivered to the cytoplasm?
Antibodies, proteins or peptides delivered with PULSin™ are only transported to the cytoplasm of the cell where it is released from the PULSin™/protein complexes. For nuclear targeting, the proteins need to have a nuclear localization signal (NLS).
I have stored PULSin™ at -20°C for one week upon receiving the tube. Can I still use it?
PULSin™ should NOT BE FROZEN as indicated on the box, therefore it is not possible to guarantee that the product is efficient anymore. You may check if PULSin™ solution is still clear and that the product has not precipitated. Moreover, the control protein RPE included in the kit could be damage.
Can PULSin™ be used for in vivo applications ?
You may try it, although the reagent was not made for such application, preliminary data from our R&D shows successful R-PE delivery into 30% of the monocyte/macrophages from ascites upon intraperitoneal delivery of 2 ml containing 50 µg of protein and 150 µl of PULSin in a total volume of 2 ml of glucose 5%.
Which type of oligodeoxynucleotides (ODNs) can be transfected with jetPEI™?
jetPEI™ successfully transfects regular phosphodiester ODN as well as any partially or fully modified phosphorothioate ODN. It has been shown to form stable complexes with ODNs protecting them from enzymatic degradation. However, jetPEI™ is unable to complex with neutral ODN such as PNA (peptides nucleic acid) or fully modified methylphosphonate.
What is the best reagent for miRNA transfection?
miRNA transfection is actually not an easy question! Several types of molecules are abusively called miRNA. The term miRNA can refer to:
- miRNA duplexes (also known as mimic miRNA). These are structurally similar but not identical to siRNA and can be transfected with INTERFERin™.
- plasmid encoding for miRNA. This can be transfected using jetPRIME™.
- pre-miRNA or antimiR can be transfected using INTERFERin™.
