Frequently Asked Questions
As the first step, we suggest consulting the ‘Transfection Reagent Selection Guide’ to find out if the cells of interest have been previously tested. If your cell type is not on the list, we suggest using the reagents optimized for similar cells (i.e., attachment-dependent/suspension or established cell line/primary cells), or contact us by e-mail for an informed suggestion.
We recommend small scale preliminary experiments to optimize the performance of our transfection reagents. As with all transfection reagents, the complex formation, cell seeding density, and culture and incubation conditions will affect the final performance. Please consult our technical sheet on optimizing transfection for this purpose (link).
The general protocols for transfecting adherent and suspension cells can be found here.
For product specific protocols, please see the following links.
Our transfection reagents are compatible with a wide range of serum-free media, including DMEM, RPMI and MEM, for preparing nucleic acid complexes. Once the complexes are prepared in serum free medium, they can be added to serum-containing medium for transfection.
The recommended nucleic acid to transfection reagent ratio ranges from (w/w) 1:1 for relatively toxic reagents to 5-20:1 for biocompatible reagents. This ratio should be optimized for each application. For suggested ranges for each reagent, please consult the specific reagent manual.
No. It is not necessary to remove the complexes from treated cells. Complexes can be left in culture with cells until end-point analysis.
Depending on the application, incubation times may vary from 2 hours to 24 hours. It may be possible to centrifuge the treated cells to accelerate the transfection process and minimize the complex incubation times.
Transfection efficiency can be determined by different approaches. Reporter genes, such as GFP or RFP, are convenient ways to assess transfection by using microscopy or flow cytometry techniques. Direct assessment of the induced gene product (e.g., by ELISA, western blot), or silenced gene expression (e.g., protein levels or mRNA levels by PCR) are important. One can also use functional outcomes as an indirect measure of transfection, although care must be paid to complicating factors in this case. In all studies, we recommend employing a control (i.e., non-active) agent similar in nature to the nucleic acid being investigated.
A methodical analysis of the factors contributing to transfection, as outlined in the ‘Considerations for optimizing non-viral transfections in culture‘, is a good place to start. Other resources to improve transfection efficiencies can be found in our ‘suggested reading’ tab. We are here to help as well, so do send us a message/e-mail to see how we can assist you.
All transfection reagents display a certain extent of cytotoxicity on cells, depending on the amount used. The key is to achieve transfection without disrupting the physiology of the cells significantly. One can minimize exposure time to transfection complexes, speed up the transfection process by centrifugation and optimize reagent/nucleic acid concentrations to eliminate unnecessary exposure. The purity of the nucleic acid is also important to eliminate unforeseen toxicities.
These are important criteria that affect the transfection efficiency. In general, transfection efficiency decreases with increased cell density and passage number, as cells settle in senescence. For other factors affecting transfection efficiency, please consult ‘Considerations for optimizing non-viral transfections in culture‘.
The recommend storage temperature is at 4 degC (short term) or -20 degC (long term). The reagents are designed to be stable for 1 year under these conditions.
Yes. Our in vivo transfection reagents display broad activities so that they could be effective under in vitro conditions.
It is likely for our transfection reagents to work with different nucleic acids. This is not universally applicable, but most reagents seem to handle different nucleic acids. ALL-Fect transfection reagent can handle both DNA and RNA.
Our reagents are based on an optimal balance of cationic charge and hydrophobicity. They are polymeric in nature that interact with nucleic acids via multivalent interactions. These reagents provide effective condensation of anionic charge of nucleic acids, while displaying little toxicity on mammalian cells.
To indicate the source of the transfection reagent, you can state the reagent name and that it was obtained from RJH Biosciences Inc. (Edmonton, AB, Canada).