This page has some useful references and resources to help you succeed. Including optimization of transfection protocols, transfection reagent selection guidance for your cells, and frequently asked questions. If you require any further assistance please do not hesitate to contact us and our team will be happy to help!
The table below summarizes optimal transfection reagents for nucleic acids in different cell types.
The efficiency of the transfection reagents was assessed by using plasmid DNA (pDNA), short interfering RNA (siRNA), and messenger RNA (mRNA).
Where the transfection reagent was suitable for both pDNA or siRNA delivery, it was indicated with pDNA/siRNA.
|Primary Cells||Umbilical Cord Blood Derived Mesenchymal Stem Cells (UCB-MSC)||pDNA||pDNA mRNA||pDNA|
|Bone Marrow Derived Mesenchymal Stem Cells (BM-MSC)||pDNA||pDNA mRNA||pDNA|
|Vascular smooth muscle Cells (VSMCs)||pDNA mRNA||pDNA|
|Human Umbilical Vein Endothelial Cells (HUVECs)||pDNA mRNA|
|Mononuclear Cells from CML patients (MNC)||pDNA mRNA||siRNA||siRNA|
|Human Foreskin Fibroblast Cells||pDNA|
|Rat Primary Sympathetic Neurons||pDNA||mRNA|
|Kidney Fibroblast Cells (293-T)||Kidney Fibroblast Cells (293-T)||pDNA||pDNA mRNA||pDNA|
|Breast Cancer Cells (MDA-MB-231)||pDNA siRNA co-delivery||pDNA mRNA||pDNA||siRNA||mRNA|
|Kidney Epithelial Cells (MDCK)||siRNA|
|Breast Cancer/Melanoma Cells (MDA-MB-436)||pDNA mRNA||siRNA||mRNA||RNP|
|Breast Cancer Cells (MDA-MB-468)||siRNA|
|Breast Cancer Cells (Sum-149PT)||pDNA mRNA||siRNA|
|Breast Cancer Cells (MCF-7)||pDNA||pDNA mRNA||pDNA siRNA||mRNA|
|Human Lymphoma Cells (U-937)||pDNA||pDNA|
|Chronic Myeloid Leukaemia Cells (K562)||siRNA||pDNA mRNA||siRNA||mRNA|
|Acute Myeloid Leukemia Cells (KG1 and KG1A)||siRNA||siRNA|
|Acute Myeloid Leukemia Cells (THP1)||siRNA||siRNA||mRNA|
|Human Lung Cancer Cells (A549)||siRNA|
|Human Colon Cancer (HCT-116)||siRNA||siRNA|
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).
Each product page has a copy their specific transfection protocol, they can be found here:
Our transfection reagents are compatible with a wide range of serum-free media, including DMEM, RPMI, MEM and others. Complexes are expected to be functional in serum-containing medium.
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 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 ‘Technical Tips to Improve Transfection’, 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 ‘Technical Tips to Improve Transfection’.
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.
We recommend to use 1:5 ratio of nucleic acid to transfection reagent, so that 1 mL vial (1 mg) is suitable for 200 transfections of 1 mg nucleic acid. However, optimal ratio may change depending on the application and cell type.
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).