RJH Biosciences is a biotechnology company based out of Edmonton, Alberta, Canada. We develop novel transfection reagents and delivery systems that transport different types of nucleic acids to a range of human cells and cell lines. We develop value-added products in two commercial segments, as transfection reagents for biomedical R&D enterprise and as nucleic acid delivery vehicles for preclinical and clinical applications. The same transfection reagents are used in our own R&D projects that focus on blood cancers and modification of immune cells.
Designed as a siRNA pDNA transfection reagent it can be used for either siRNA knockdown, plasmid DNA transformation or siRNA pDNA co-delivery. It is particularly suitable for mesenchymal stem cells from cord blood and bone marrow, and highly differentiated cells such as smooth muscle cells, and endothelial cells.
Provides 2 to 3-fold higher efficacy in the presences of serum
Simple Protocol
No need to change tissue culture medium during transfection
Toxicity
Less toxic compared to commercial transfection reagents, leading to better retention of normal cellular physiology
References:
Hsu and Uludağ. Biomaterials (2012) 33: 7834-7848.
Remant Bahadur et al., J. Materials Chemistry B (2015) 3: 3972-3982.
Wang et al., J. Surgical Research (2013) 183: 8-17.
Images: Typical performance of All-Fect for transfecting cord-blood derived mesenchymal stem cells with a GFP plasmid. The study compared the performance of All-Fect against a leading lipofection reagent under conditions optimized for each reagent. The extent of transgene expression was quantitated by flow cytometry, based on the extent of EGFP expression in arbitrary units. Typical fluorescent micrographs showing GFP expression after transfection are provided on the top.
Prime-Fect
This product is designed for transfection of primary cells with plasmid DNA. Prime-Fect has been tailored for attachment dependant cell plasmid DNA delivery, but also has been effective in siRNA delivery to attachment dependant cells and plasmid transfection of non-attachment dependant cells.
on primary cellsProvides 2 to 3-fold higher efficacy in the presences of serum
Simple Protocol
No need to change tissue culture medium during transfection
Toxicity
Less toxic compared to lipofection reagents, leading to minimal alteration of cell physiology
References:
KC et al., J. Materials Chemistry B (2015) 3: 3972-3982.
Images: Transfecting bone marrow stromal cells with Prime-Fect. GFP expression was induced with a plasmid and analyzed by fluorescent microscopy 2 days after transfection.
(A) Cell-associated DNA is visualized with a red label.Transfecting breast cancer MDA-MB-231 and MCF-7 cells with siRNA using Prime-Fect.
(B) The expression levels of two target genes were analyzed by qPCR after delivery of control (scrambled) and gene-specific siRNAs (2 days after transfection).
(C,D) Transfecting umbilical cord-derived mesenchymal stem cells with Prime-Fect. GFP expression was induced with a plasmid DNA and analyzed by fluorescent microscopy 2 days after transfection.(C) A leading polymeric transfection reagent and (D) Prime-Fect.
Leu-Fect A & B
These products are designed for transfection of attachment-independent (suspension growing) cells and are specifically tailored for siRNA delivery. We offer two different formulations (Leu-Fect A and Leu Fect B) that are effective in different types of attachment-independent cells. We recommend the users to test the efficacy of both reagents in their particular cell type and choose the right formulation for long-term use.
Provides 2 to 3-fold higher efficacy in the presences of serum
Simple Protocol
No need to change tissue culture medium during transfection
Toxicity
Less toxic compared to lipofection reagents, leading to minimal alteration of cell physiology
References:
Gul-Uludag et al. Leukemia Research (2014) 38: 1299–1308.
Landry et al. J. Controlled Release (2016) 224: 8-21.
Valencia-Serna et al. J. Controlled Release (2013) 172: 495-503.
Images: Uptake of Leu-Fect/FAM-siRNA complexes in K562 cells in vitro (A) and in vivo K562 tumors (B). Typical performance of Leu-Fect for transfecting K562 chronic myeloid leukemia (CML) cells with a specific siRNA against Bcr-Abl.
The study compared the performance of Leu-Fect against a leading lipofection reagent and branched PEI (PEI25; 25 kDa) under conditions optimized for each reagent. The extent of gene silencing was quantitated by qPCR (C). The functional outcome, in the form of inhibition of cell growth, was assessed by the MTT Assay and expressed relative to non-treated cells (D).
Trans-Booster
This product is designed to enhance the transfection efficiency of plasmid DNA in attachment dependent and suspension-growing cells. Enhancing the transfection efficiency allows production of greater amount of protein for a desired effect, as well as allowing lower amount of DNA and transfection reagent to be used. This reagent could be also effective with other nucleic acids (siRNA, microRNA) depending on the cell type.
Provides 2 to 3-fold higher efficacy in the presences of usual transfection reagent
Simple Protocol
No need to change tissue culture medium during transfection
Toxicity
Less toxic compared to lipofection and polymeric reagents, leading to better protein yields from the transfected cells
Stability
Long term stability at room temperature
Figure: Typical performance of Trans-Booster reagent for transfecting suspension growing Jurkat cells (top). Transfections with a GFP plasmid and a GFP mRNA are conducted in Jurkat cells with and without Trans-Booster in the transfection formulation. A leading lipofection reagent was compared to the All-Fect transfection reagent from RJH Biosciences in this study. The extent of transgene expression was quantitated by flow cytometry and summarized as the mean GFP fluorescence in arbitrary units. Note that for both mRNA and plasmid DNA based GFP expression, Trans-Booster gave a significant increase in transfection efficiency.
In Vivo DNA-Fect
This product is designed for delivery of plasmid DNA in animal models. It is a cost-effective alternative to PEI-based reagents marketed for in vivo applications, with improved transfection efficiency. The application of current reagents is usually limited by their toxicity, which either limits the expression of the transgenes or alters the physiological response in the vicinity of the administered agents. The biocompatibility of In Vivo DNA-Fect minimally alters the cellular physiology, allowing a better response of the expressed transgenes.
Provides 2 to 3-fold higher efficacy under serum conditions
Simple Protocol
Can be administered with simple formulations such as saline
Toxicity
Less toxic compared to commercial transfecting reagents, leading to better protein yields from the transfected cells
References:
Rose et al., Biomaterials Science (2014) 2: 833-842.
Rose et al., Biomaterials (2012) 33: 3363-3374.
Images: Typical performance of In Vivo DNA-Fect for transfecting tissues with two separate GFP plasmids at the rat subcutaneous site is shown in the figure. The study compared the performance of In Vivo DNA-Fect against branched PEI (PEI25; 25 kDa), after the corresponding complexes were implanted in gelatin sponges for 14 days. The extent of transgene expression was assessed by histology of explanted tissues, followed by the fluorescent microscopy.
In Vivo RNA-Fect
This product is designed for delivery of siRNA in mice tumor models. It can be directly administered into a specific site (e.g. intratumorally), or systematically (e.g. intraperitoneal, intravenous). It is a cost-effective alternative to PEI-based reagents marketed for in vivo applications. It’s low toxicity
allows for higher doses of nucleic acids to be administered without affecting the health of animal models.
Provides effective transfection under physiological conditions
Simple Protocol
siRNA particles can be prepared in saline suitable for administration
Toxicity
Less toxic compared to other commercial transfecting reagents
References:
Aliabadi et al., J. Controlled Release (2013) 172: 219-228.
Alshamsan et al., Translational Oncology (2011) 4: 178-188.
Abbasi et al., Pharmaceutical Research (2011) 28: 2516-2529.
Images: Tumor growth profiles as a result of siRNA delivery with In Vivo RNA-Fect is shown in the figure. The tumors were left untreated, or treated with scrambled and therapeutic siRNAs formulated with the transfection reagent. Formulations injected intratumorally (A). Formulations injected intraperitoneally (B).
mRNA-Fect
This product is designed for mRNA delivery of both attachment-dependent and attachment-independent (suspension growing) cells. mRNA-Fect has also been tested and found effective for plasmid DNA and siRNA delivery in certain cell types. We recommend the users to test the efficacy of the reagent in their particular cell type and choose the right formulation for long-term use.
Provides 2 to 7-fold higher efficacy in the presences of serum compared with lipofection reagents
Simple Protocol
No need to change tissue culture medium during transfection
Toxicity
Less toxic compared to lipofection reagents, leading to minimal alteration of cell physiology
Images: Transfection of mRNA with mRNA-Fect in (A) attachment-dependent MCF-7, MDA-MB-436 and MDA-MB-231 cells and (B) suspension-growing K562 and THP-1 cells. An mRNA coding for a reporter protein (Green Fluorescent Protein, GFP) was used to assess the efficiency of mRNA expression. Typical GFP expression levels were visualized under fluorescent microscopy (top pictures). The expression levels were quantitated by flow cytometry 72 hours after transfection and summarized as the percentage of cells positive for GFP (bottom graphs). For comparison, a leading lipofection reagent was used according to the manufacturer’s instructions.
CRISP-Fect
This product is designed for CRISPR-Cas9 RNP delivery to various attachment dependent and suspension cell types. Please click on the “view product” link below for information and example data on applied cell types.
We recommend that users test the efficacy of the reagent in their particular cell type and choose the right formulation for long-term use. General optimization protocols can be found here. Applied cell types for all of our commercial reagents can also be found on the resources page. Specific protocols can be found in the following brochure below.
Provides 2 to 7-fold higher efficacy in the presences of serum compared with lipofection reagents
Simple Protocol
No need to change tissue culture medium during transfection
Toxicity
Less toxic compared to lipofection reagents, leading to minimal alteration of cell physiology
Figures: Left. Transfection of Jurkat T-cells and breast cancer MDA-MB-436 cells using CRISP-Fect. sgRNA/Cas9 complexes (RNP) were formulated with indicated transfection reagents and added to the cells for 24 hours. RNP uptake was followed by using FITC-labeled Cas9. The results are summarized as arbitrary fluorescence units, indicating the average amount of RNPs delivered per cell. Right. Transfection of Jurkat T-cells with RNPs formulated at different ratios of Cas9/sgRNA using CRISP-Fect. Lipofectamine 2000 and jetOptimus was used as commercial reference reagents in these studies.
Feature of Products
We developed broadly acting transfection reagents to modify mammalian cells with plasmid DNA, siRNA, mRNA and other nucleic acids. The foundation of our transfection reagents is based on cationic lipopolymers with optimal balance of cationic charge and hydrophobic (lipid) group. By systematically altering the polymeric backbone and the nature of lipid group, a library of transfection reagents has been generated. Some of the transfection reagents are broadly acting, functioning in different cell types with different nucleic acids. Others display high specificity, where they are exceptionally effective in particular cell types for particular nucleic acid delivery.
The key advantages of our delivery vehicles are:
Multivalent interactions with nucleic acids leading to strong binding of cargo that withstand disruptive forces in transit through cell membranes.
Synergistic effects due to cationic and lipidic binding that coat the cargo and protect it from nucleases.
Lipidic moieties that enhance interactions with cell memblane and internalization.
pH buffering capacitythat facilitate escape of cargo from endosomes.
Tailored formulations to free nucleic acids once internalized in cytoplasm.
The transfection reagents have been optimized for the following cell models and applications:
One area of focus for RJH Biosciences is to implement RNA interference (RNAi) via delivery of short interfering RNA (siRNA). Our initial therapeutic application is blood cancers, while recognizing that the RNAi activity can be implemented in the treatment of a large range of human cancers and other diseases. Another focus is direct administration of plasmid DNA (pDNA) to express therapeutic proteins in situ, with applications in immunotherapy.
Why study blood cancers and immunotherapies with nucleic acid therapeutics?
There are three types of blood cancers: Leukemia, lymphoma, and myeloma. Leukemia is characterized by highly proliferating, abnormal white blood cells [1]. Lymphoma and myeloma are respectively cancers of the lymphatic system and plasma cells which greatly effect the immune system [2,3]. These three cancers are difficult to treat and the current treatments are limited in efficacy, especially at the end stage of the disease.
The use of nucleic acid-based therapeutics can eradicate these cancers in two primary ways, with RNAi technology and cell-based immunotherapy. The use of RNAi is being increasingly explored in the treatment of the blood cancers. Polynucleotides such as siRNA has aided the downregulation of oncogenes and can be designed to support specific abnormalities in individual patients, making it a ‘personal’ strategy with a universal technological design [4]. Due to siRNA’s potential in blood cancer therapies, we are currently focusing on siRNA therapeutics in our R&D projects, targeting disease-driving oncogenes and inducing apoptosis in the malignant cells.
Another strategy that is being explored for treating blood cancers is the use of immunotherapy. Immunotherapeutic strategies include the use of antibodies, stem cell transplants, cytokines, small molecules among others [5]. However, a more recent approach is genetic therapy by using engineered cells, also known as Cell Transfer Therapy. This method works by taking patients’ own immune cells such as but not limited to T-cells, B-cells, and NK cells. The immune cells genome is engineered to support various therapeutic strategies that may involve neoantigen expression and presentation on immune cell surface, and then are reintroduced into the host [5]. The modified cells are ultimately designed to target and remove the malignant cells. This strategy is highly advantageous as T-cells can ‘seek’ and destroy the malignant cells in the blood system. While this approach has been promising in blood cancers, it can be also used in other solid cancers. As the foundation of immunotherapy relies on nucleic acid introduction into patient cells, efficient delivery of nucleic acids is imperative for success. Our transfection reagents offer the best in class vehicles to undertake such a delivery.
Nanomedicine based on nucleic acid therapeutics is a large component to personalized cancer therapies and immunotherapies. The RJH Biosciences strives to provide quality transfection reagents, whether it involves the delivery of our own nucleic acid candidates or our customers’.
Jean, C. and Dick, J. (2005) Cancer stem cells: lessons from leukemia. Trends in cell biology. 15, 494-501. Woods, N. et al. (2006) Therapueti gene causing lymphoma. Nature. 440, 1123. Mahindra, A. et al. (2012) Latest advances and current challenges in the treatment of multiple myeloma. Nature Reviews Clinical Oncology. 9, 135-143. Uludağ, H. et al. (2016) Current attempts to implement siRNA-based RNAi in leukemia models. Drug Discovery Today. 21, 1412-1420. Zou, W. (2006) Regulatory T cells, tumour immunity and immunotherapy. Nature Reviews Immunology. 6, 295-307.
Highly effective transfection reagent optimized for ribonucleoprotein (RNP) delivery to both attachment-dependent and suspension-growing cells.
0.75 ml/1.5 ml
Transfection Reagent Selection Guide
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.
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).
Please contact us for any inquiries, questions, or information requests.
Tokyo Future Style, Inc. info@tokyofuturestyle.com
TEL：029-851-9222 FAX：029-851-9220
