In side-by-side cotransfection experiments with traditional reagents developed for plasmid delivery, only DharmaFECT Duo Transfection Reagent resulted in efficient cell delivery of both siRNA (assayed by target gene knockdown) or microRNA reagents, along with plasmid (assayed by level of reporter expression).
DharmaFECT Duo is an excellent choice for experiments requiring cotransfection, such as Edit-R Gene Engineering, RNAi rescue or utilization of reporter systems.
- Effective delivery of two distinct molecules, DNA plasmid and small RNA, from a single formulation
- Efficient transfection without compromising cell viability
- Potent RNAi results together with plasmid expression
- Recommended for use with Edit-R Gene Engineering Cas9 plasmid and synthetic components
Editing of PSMD7 gene in U2OS-(Ubi)EGFP cells using Edit-R Cas9 Nuclease protein NLS delivered by DharmaFECT transfection reagents
U2OS-(Ubi)EGFP cells were plated at 10,000 cells/well in 96-well plates and co-transfected using DharmaFECT transfection reagents with 25 nM Edit-R Cas9 Nuclease protein NLS and synthetic crRNA:tracrRNA at 50 or 100 nM targeting PSMD7. Cells were harvested 72 hours post-transfection and the relative frequency of gene editing was calculated based on a DNA mismatch detection assay with T7 Endonuclease I. UT = untreated sample, MW = FastRuler Low Range DNA Ladder (Thermo Scientific).
Editing of VEGFA gene in HEK293T cells using Edit-R Cas9 Nuclease mRNA delivered by DharmaFECT transfection reagents
HEK293T cells were plated at 20,000 cells/well in 96-well plates and co-transfected using DharmaFECT transfection reagents with 200 ng of Edit-R-Cas9 mRNA and synthetic crRNA:tracrRNA targeting VEGFA. Cells were harvested 72 hours post-transfection and the relative frequency of gene editing was calculated based on a DNA mismatch detection assay with T7 Endonuclease I. DF1 = DharmaFECT 1, Duo = DharmaFECT Duo, UT = untreated sample, MW = FastRuler Low Range DNA Ladder (Thermo Scientific).
DharmaFECT Duo is highly effective in co-transfection of plasmid with siRNA or microRNA
DharmaFECT Duo transfection reagent achieves reliable cotransfection results for a variety of experimental goals. The graph demonstrates specific modulation of Renilla luciferase (Rluc) gene:
Rluc expression is similar for Plasmid Alone and Plasmid+Control
Down-regulation of the Rluc as a result of miRNA mimic (plasmid + miRNA mimic)
Excellent knockdown of the Rluc by siRNA (Plasmid + siRNA)
The psiCHECK-2 vector (100 ng/well; Promega) with cloned microRNA recognition sites for miR-28, miR-95, miR-30d, and miR-105, respectively, were complexed alone or with RNAi reagents (10 nM) using DharmaFECT Duo (0.2 μL/well) in MCF-7 cells at 10,000 cells/well (96-well plate). The RNAi reagents were Dharmacon miRIDIAN Mimics (Negative Control, miR-28, miR-95, miR-30d, and miR-105, respectively) or siRNA (Renilla luciferase pool). Firefly and Renilla luciferase expression was assessed at 48 hours using the Dual-Glo Luciferase Assay System (Promega) and normalized to identically treated psiCHECK-2 empty vector.
- X. Guanlan et al., Preventing β-Cell Loss and Diabetes With Calcium Channel Blockers. Diabetes. 61(4), 848-856 (Apr 2012).
- Y. Wang et al., Suppression of GSK-3β activation by M-cadherin protects myoblasts against mitochondria-associated apoptosis during myogenic differentiation. J. Cell Sci. 124, 3835-3847 (November 2011).
- X. Wang et al., Characterization of Promoter Elements Regulating the Expression of the Human Neurotensin/Neuromedin N Gene. J. Biol. Chem. 286(1), 542-554 (7 January 2011).
- S. Sinha et al., Von Hippel-Lindau Gene Product Modulates TIS11B Expression in Renal Cell Carcinoma: Impact On Vascular Endothelial Growth Factor Expression In Hypoxia. J. Biol. Chem. 284(47), 32610-32618 (20 November 2009).
- A. Poleshko et al., Identification of Cellular Proteins That Maintain Retroviral Epigenetic Silencing: Evidence for an Antiviral Response. J. Virol. 82(5), 2313-2323 (March 2008).