My 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 jetPRIME® is increased when used in the presence of serum. The complexes can therefore be added directly to the cells in serum-containing medium.
What does the DNA/jetPRIME® ratio mean?
The ratio gives the number of microliters of jetPRIME® to use per microgram of DNA. For example, a 1:2 DNA to jetPRIME® ratio means 2 µl jetPRIME® per µg DNA.
Can I use antibiotics during jetPRIME®-mediated transfection?
jetPRIME® transfection efficiency is not affected by the presence of antibiotics. The routine validation protocol for each new batch of jetPRIME® is always performed in serum and antibiotics containing medium.
How can I improve transfection efficiency?
The first step of optimization is to increase the DNA amount up to 1.5 fold. We also recommend increasing the DNA to jetPRIME® ratio (2 to 3 µl jetPRIME® per µg DNA). Another method consists in gentle centrifugation of the culture plate (5 min at 210 g).
Can I play around with cell density?
Cell density as well as passage numbers will affect transfection performance. For optimal transfection with jetPRIME®, we recommend transfecting cells at 60% to 80% confluency. Please refer to Table 1 in the jetPRIME® transfection protocol for the recommended number of cells to seed according to the cell culture plate. In addition, if cells have been in culture for a long time (over 20 cell passages), we recommend starting a new culture with a fresh tube of cells from liquid nitrogen.
Can I use the same conditions for every cell line?
The protocol provided with jetPRIME® has been optimized for a large variety of cell lines such as A549, MCF7, U-2 OS, NIH-3T3, B16-F10, Caco-2… However specific protocols have been developed for specific cell lines in order to achieve a higher efficiency. These specific conditions can be viewed in the Polyplus Cell transfection Database.
HEK-293 and HeLa cells: Are there any specific recommendations using jetPRIME® onto HEK-293 and HeLa cells?
jetPRIME® is very efficient for DNA transfection onto HEK-293 and HeLa cells. Hence, we recommend decreasing the DNA amount (from 1 µg to 0.5 µg per well for a 6 well plate for example), and using a 1:2 DNA to jetPRIME® ratio.
How do I scale up a transfection experiment with jetPRIME®?
In general, the amount of DNA should be proportional to the total number of cells. Refer to the regular jetPRIME® protocol indicating the amount of DNA and jetPRIME® needed according to the size of culture dish used (Table 2).
How should I proceed when I want to co-transfect several plasmids in the same experiment?
For cotransfection of multiple plasmids, the total DNA amount per well should not exceed the DNA amount indicated in the protocol. The ratio to use for each plasmid depends on the size of the plasmid, the plasmid construct and the expected expression level (of each plasmid). Each plasmid should represent at least 10% of the total DNA amount per well.
How long are the jetPRIME®/DNA complexes stable for?
Optimal transfection efficiency is obtained with complexes formed for 15 to 30 minutes. Hence, for High Throughput Screening applications requiring longer incubation times, we recommend using jetPEI® for HTS as jetPEI®/DNA complexes are stable up to 4 hours.
Is jetPRIME® convenient for virus production?
jetPRIME® is the reagent of choice for virus production in classical medium as detailed here. Indeed, transfection efficiency using jetPRIME® reaches up to 90% in cells commonly used for virus production, such as HEK-293 and derivatives, CHO, VERO, WOP and BHK cells, hence leading to high viral titers (Dewannieux, M., Vernochet, C., Ribet, D., Bartosch, B., Cosset, F. L., Heidmann, T. (2011). The mouse IAPE endogenous retrovirus can infect cells through any of the five GPI-anchored Ephrin A proteins., PLoS Pathog 7, e1002).
How to improve cell viability of fragile cells (e.g. primary fibroblasts)?
Transfection is quite a traumatic event for the cells. In order to improve cell viability, find below several suggestions:
- change the medium 4h after transfection
- decrease the DNA amount
- decrease the volume of jetPRIME®
- perform transfection in serum-containing medium
- analyze the transfection efficiency 24 h after transfection after transfection instead of 48 h
- ensure that jetPRIME® and DNA are diluted into the jetPRIME® buffer
- check that the plasmid is endotoxin free
Is there a plasmid size restriction for DNA transfection using jetPRIME®?
There is no size restriction for DNA transfection using jetPRIME®. However, keep in mind that for the same amount of DNA, the gene copy number will be less for large DNA than for smaller plasmids.
Could I transfect oligonucleotides using jetPRIME®?
jetPRIME® is not the reagent of choice for ODN transfection. We usually recommend using INTERFERin® or jetPEI®, depending on the size of the oligonucleotides. Please contact the technical support if you wish to perform ODN transfection (contact us).
Can I transfect siRNA using jetPRIME®?
jetPRIME® is perfectly suitable for siRNA transfection, using 10 to 50 nM siRNA final concentration. However, if you are mostly interested in siRNA transfection, then INTERFERin® is the reagent of choice.
What type of compound is jetPRIME®?
jetPRIME® is a novel proprietary cationic polymer-based molecule, synthesized at Polyplus-transfection.
How is the plasmid delivered when transfection is performed with jetPRIME®?
JetPRIME® forms positively charged complexes with DNA. These complexes then penetrate the cell through endocytosis. The endosomes then release DNA in the cytosol via the proton sponge mechanism. The plasmid mostly reaches the nucleus when the nuclear envelope disappears during mitosis.
How should jetPRIME® be stored?
jetPRIME® is a very stable molecule. jetPRIME® and its buffer are shipped at room temperature and should be stored at 4°C to ensure long term stability.
When I received my jetPRIME® vial, it was completely warm. Is it still efficient for further use?
jetPRIME® is a very stable molecule. Experiments were conducted in house to test the stability of the jetPRIME® reagent. After 48 hours at 50°C, jetPRIME® shows a similar transfection efficiency as if stored at 4°C.
I have accidentally frozen my jetPRIME® vial. What should I do?
jetPRIME® can undergo an accidental freeze/thaw cycle without compromising the transfection efficiency. However, for long term storage we recommend aliquoting and storing the reagent at 4°C to ensure long term stability.
How long can I use my jetPRIME® vial for?
jetPRIME® cat#114-01 (0.1 ml) is stable for 6 months. Other pack sizes of jetPRIME® are stable for at least one year, when stored appropriately.
Where could I find publications from researchers who have successfully used jetPRIME®?
Scientific papers in which jetPRIME® as well as other Polyplus-transfection reagents are cited can be found in the Polyplus Product Citation Database .
Do you have any specific conditions for given cell lines?
Please feel free to browse the Polyplus Cell Transfection Database, in which specific conditions for many cells may be found: http://www.polyplus-transfection.com/technical-resources/cell-line-database/
Is jetPRIME® recommended for DNA transfection into suspension cells?
Suspension cells, such as T and B lymphocytes, are notoriously hard to transfect cells with DNA, whatever the chemical reagent used. Hence we usually recommend using electroporation or virus transduction for nucleic acid delivery into suspension cells.
Has jetPRIME® been already tested for transfection into vegetal cells?
Vegetal cells have a cellulose rich-membrane that prevents jetPRIME®/DNA complexes entering the cells. Even at the protoplasts stage where the cellulose envelope has been removed, complexes are not able to penetrate into the cytoplasm.
Is jetPRIME® efficient for DNA transfection onto HUVEC?
For HUVEC cells, we recommend using jetPEI®-HUVEC, a reagent specifically designed for this purpose as described here.
Can I use jetPRIME® on primary neurons culture?
Neurons are very difficult to transfect mainly because they do not divide in culture. However, jetPEI® has been successfully used for the transfection of neurons in vitro. 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 jetPRIME® for DNA transfection into macrophages?
Raw264.7 are successfully transfected with DNA using jetPRIME®. A transfection efficiency of 50% can be achieved following the standard conditions. For DNA transfection onto primary macrophages or monocyte/dendritic cell-derived macrophages, jetPEI®-macrophage is the reagent of choice. Please click here for more information in a wide variety of cells.
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.
Which type of cell lines has INTERFERin® already been successfully tested on?
INTERFERin® has been successfully used on various adherent and non-adherent cell lines such as HeLa, HEK-293, A549, MDA-MB-231, BHK-21, CHO and OVCAR-3. 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 Polyplus Cell Transfection Database.
Do you recommend using INTERFERin® for miRNA transfection?
Several types of molecules are abusively called miRNA. The term miRNA can refer to:
- 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®.
- antimiR: this is a single-stranded nucleic acid designed to specifically bind to and inhibit endogenous microRNA; it can be transfected using INTERFERin®.
Can I use INTERFERin® for in vivo applications?
INTERFERin® is dedicated only to in vitro transfections. For siRNA delivery into animals, we recommend using in vivo-jetPEI®, which has been successfully used for siRNA and oligonucleotides delivery into a wide panel of animals. Please to the in vivo-jetPEI® web page for more information.
How should INTERFERin® be stored?
INTERFERin® should be stored tightly capped at 4°C.
I accidentally froze my vial of INTERFERin® at -20°C. Is is still efficient for further use?
INTERFERin® should not be frozen, as this strongly compromises its efficiency. We recommend discarding the frozen tube and using a new one.
How long can INTERFERin® be kept?
INTERFERin® cat#409-01 (0.1 ml) is stable for 6 months. Other pack sizes of INTERFERin® are stable for at least one year, when stored appropriately
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.
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 exhaustive list please refer to the Polyplus Cell Transfection Database.
Which proteins have been delivered 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 accidentally froze my vial of PULSin® at -20°C. Is it still efficient for further use?
PULSin® should not be frozen, as this strongly compromises its efficiency. We recommend discarding the frozen tube and using a new one.
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).
How should I store in vivo-jetPEI®?
in vivo-jetPEI® is very stable and shipped at room temperature. However, for long-term conservation, we recommend aliquoting in vivo-jetPEI® upon reception and then freezing at -20°C.
What kind of nucleic acids can be delivered with in vivo-jetPEI®?
Different types of nucleic acids have been successfully delivered using in vivo-jetPEI®: DNA, siRNA, mimic miRNA, antimiR, oligonucleotides…Please refer to the Polyplus Product Citation Database for more details.
In which animals can in vivo-jetPEI® be used?
In vivo-jetPEI® has been tested in many species such as mice, rats, mosquitoes, shrimps, guinea pig, macaques… Please feel to contact the technical support for more information (contact us).
Which administration routes can be used for in vivo-jetPEI®-mediated nucleic acid delivery?
In vivo-jetPEI® is suitable for systemic and local nucleic acid delivery. Several ways of administration have been successfully tested such as intravenous injection (tail vein and retroorbital), intraperitoneal injection, intratumoral injection, intracerebral injection, intranasal instillation, as detailed here.
I would like to target the lung. What is the best injection route?
In vivo-jetPEI® is the reagent of choice for nucleic acid delivery to the lung. Indeed, the lung is the most targeted organ following systemic delivery of in vivo-jetPEI®/nucleic acid complexes.
I want to target a specific organ; how should I proceed?
The delivery procedure highly depends on the organ to target. Depending on the route of administration, in vivo-jetPEI®-mediated gene expression was observed in the brain, liver, pancreas, spleen, kidney, heart, bladder, skin, retina, etc. Please contact our technical support team for more advice (contact us).
What is the best reagent for DNA HTS applications?
jetPEI® transfection reagent is particularly well suited for automated or manual HTS with three protocols available: reverse, batch and forward. jetPEI® provides highly reproducible results and the DNA/jetPEI® complexes are stable for up to 4 hours. Please click here for more information.
What is the best reagent for siRNA HTS applications?
INTERFERin®-HTS is a new generation siRNA transfection reagent especially developed for HTS applications providing great silencing efficiency, excellent reproducibility and high cell viability with very low amounts of reagent. INTERFERin®-HTS is cost-effective, easy to handle, compatible with serum and antibiotics, and comes with reverse and forward protocols for 96- and 384-well plates, as described here.
Can I use INTERFERin®-HTS for standard siRNA transfections?
INTERFERin®-HTS is not the reagent of choice for standard siRNA applications. Please choose INTERFERin® siRNA Tranfection Reagent for day-to-day transfections.
What is the molecular weight of 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.
Can I use jetPEI® for other applications than HTS?
jetPEI® can be used for other applications than HTS such as DNA delivery in cells grown in suspension. In addition Polyplus-transfection developed a specific protocol for jetPEI®-mediated DNA transfection into primary neurons. Please contact the technical support team for more information (contact us).