Transfection

Reagents optimized for your specific application

Dharmacon transfection reagents are designed specifically for small RNA transfection (DharmaFECT), co-transfection of plasmid and small RNA (DharmaFECT Duo), plasmid transfection (DharmaFECT kb) and self delivering siRNA for difficult to transfect cell types (Accell siRNA).

One of the key success factors in any gene modulation experiment (RNAi, overexpression, gene editing) is the introduction of RNA and/or DNA components into your cell line or in vivo system. When applying lipid-based transfection reagents, there are three general measures of an effective transfection reagent:

  • High efficiency delivery
  • Low cellular toxicity
  • Broad dynamic range

We offer a variety of Dharmacon DharmaFECT transfection reagents with demonstrated success for all of these key criteria. Not only do we specialize in transfection of small RNA (siRNA, microRNA, crRNA, tracrRNA) but we offer DharmaFECT Duo, specifically designed for co-delivery of small RNA with plasmid DNA, and DharmaFECT kb for delivery of plasmid DNA.

  • DharmaFECT 1 Transfection Reagent

    The most broadly applicable DharmaFECT formulation for optimal siRNA or microRNA transfection into a wide range of cell types for successful RNAi experiments. DharmaFECT 1 has been validated in over 35 cell types.

  • DharmaFECT 2 Transfection Reagent

    One of four siRNA/microRNA specific formulations, DharmaFECT 2 is a chemically distinct alternative to one-size-fits-all transfection reagents to achieve high-efficiency silencing in more cell types.

  • DharmaFECT 3 Transfection Reagent

    One of four siRNA/microRNA specific formulations, DharmaFECT 3 is a chemically distinct alternative to one-size-fits-all transfection reagents to achieve high-efficiency silencing in more cell types.

  • DharmaFECT 4 Transfection Reagent

    DharmaFECT 4 Transfection Reagent is chemically distinct from the other DharmaFECT formulations, providing an alternative when DharmaFECT 1 does not achieve optimal results, and is ideal to include in transfection optimization experiments prior to RNAi screening or other high-value experiments

  • DharmaFECT Set of 4 Transfection Reagents

    An aliquot of each of the four DharmaFECT formulations for siRNA/microRNA transfection optimization studies. Useful for determination of the best reagent for your cells and particular experimental conditions.

  • DharmaFECT Duo Transfection Reagent

    For high-confidence co-transfection

  • DharmaFECT kb Transfection Reagent

    Efficient transfection of plasmid DNA with minimal cytotoxicity

  • Accell siRNA Reagents

    Self-delivering siRNA for targeted gene silencing in difficult-to-transfect cells

Choose from positive control siRNAs or our novel transfection indicators

siGLO Positive Control Reagents Species Catalog Number
siGLO Cyclophilin B Control siRNA Human, Mouse, Rat D-001610-01
siGLO Lamin A/C siRNA Human, Mouse, Rat D-001620-01
siGLO Lamin A/C Control siRNA H D-001620-02
siGLO Lamin A/C Control siRNA M D-001620-03
siGLO Lamin A/C Control siRNA R D-001620-04
siGLO Negative Control Reagents Species Catalog Number
siGLO RISC-Free Control Human, Mouse, Rat D-001600-01
siGLO Transfection Indicators Species Catalog Number
siGLO Green Transfection Indicator Human, Mouse, Rat D-001630-01
siGLO Red Transfection Indicator Human, Mouse, Rat D-001630-02

DharmaFECT Transfection Reagent

The table below lists transfection recommendations to help you select the appropriate DharmaFECT formulation for your research. You can also download a PDF version of the DharmaFECT Cell Type Guide here.

All experimental conditions resulted in cell viability and positive control gene silencing of 80% or better.
All experiments were done in 96-well plates with Non-targeting control siRNA and PPIB (Cyclophilin B) or GAPD Control pools at 25 nM; Alamar Blue (viability) and knockdown measured at 24 hours.
Data normalized to untransfected for viability and both untransfected and Non-targeting control for knockdown.
Transfection conditions should always be re-evaluated in the context of a new plate format or assay-specific requirements for cell density.
Human
Cell line Cell type Recommended DharmaFECT formulation DharmaFECT volume/well (μL) in 96 well plate Plating density in 96 well plate (cells per well) Other successful formulations
786-0 Kidney adenocarcinoma 1 0.4 5 × 103 2
A549 Lung carcinoma 1 0.2 1 × 104 2, 3, 4
ARPE-19 Retinal pigment epithelial 4 0.2 1 × 104 1,2
BxPC3 Pancreas adenocarcinoma 2 0.2 5 × 103 1, 3, 4
DLD-1 Colorectal adenocarcinoma 2 0.4 5 × 103 1,3
DU 145 Prostate carcinoma 1 0.2 1 × 104 2, 3, 4
NCI-H1299 Lung carcinoma 2 0.2 1 × 104 4
HCT-116 Colorectal carcinoma 2 0.1 5 × 103 4
HEK293 Kidney transformed embryonic cells 1 0.2 1 × 104 2, 4
HeLa Cervical epithelial adenocarcinoma 1 0.2 5 × 103 2, 3, 4
HeLa S3 Cervical epithelial adenocarcinoma 4 0.4 5 × 103 1, 2, 3
Hep G2 Hepatocellular carcinoma 4 0.4 1 × 104 1, 2
hMSC Mesenchymal stem cells 1 0.4 5 × 103 2, 3, 4
HT-29 Colorectal carcinoma 1 0.2 5 × 103 2, 3, 4
HT1080 Fibrosarcoma 4 0.2 5 × 103 1, 2, 3
Huh7 Hepatocarcinoma 4 0.05 5 × 103 1, 2
HUVEC Umbilical vein endothelial cells 4 0.2 2 × 104 1, 2
JEG-3 Placenta, choriocarcinoma, epithelial 3 0.2 1 × 104  
LNCaP Prostate carcinoma 3 0.2 1 × 104 1
MCF Breast adenocarcinoma 1 0.2 1 × 104 2, 4
MCF 10A Breast adenocarcinoma 1 0.2 1 × 104 2
MDA-MB-453 Breast adenocarcinoma 2 0.2 1 × 104 1, 3, 4
MDA-MB-231 Breast adenocarcinoma 4 0.1 5 × 103 1
OVCAR3 Ovarian adenocarcinoma 1 0.1 5 × 103 2, 3, 4
PC-3 Prostate carcinoma 2 0.2 1 × 104 3
Saos-2 Bone, osteosarcoma, epithelial 1 0.05 1 × 104 2,3,4
SK-BR-3 Breast adenocarcinoma 2 0.2 1 × 104 1, 3, 4
SKOV-3 Ovarian adenocarcinoma 3 0.4 1 × 104 1, 2, 4
U-87 MG Brain glioblastoma 1 0.1 5 × 103 2, 3, 4
Rodent
Cell line Cell type Recommended DharmaFECT formulation DharmaFECT volume/well (μL) in 96 well plate Plating density in 96 well plate (cells per well) Other successful formulations
A7r5 Rat aortic smooth muscle 2 0.1 5 × 103 1
C2C12 Mouse myoblasts 1 0.2 5 × 103 2, 3, 4
CHO K1 Chinese hamster ovary 4 0.8 1 × 104 1, 2
ES-D3 Mouse embryonic stem cells 1 0.2 2 × 103 2
ES-E14TG2a Mouse embryonic stem cells 1 0.2 2 × 103 2
H9c2 Rat myoblasts 1 0.2 1 × 104 2, 3, 4
J774A.1 Mouse macrophages 4 0.2 1 × 104 -
NIH/3T3 Mouse embryonic fibroblasts 1 0.2 1 × 104 3
NRK-49F Rat kidney fibroblasts 2 0.2 1 × 104 1, 4
Rat2 Rat fibroblasts 1 0.2 2 × 104 2
3T3-L1 Mouse embryonic fibroblasts 1 0.2 5 × 103 3
Other
Cell line Cell type Recommended DharmaFECT formulation DharmaFECT volume/well (μL) in 96 well plate Plating density in 96 well plate (cells per well) Other successful formulations
COS-7 African green monkey kidney 2 0.4 5 × 103 1, 3, 4

Editing of PSMD7 gene in U2OS-(Ubi)EGFP cells using Edit-R Cas9 Nuclease protein NLS delivered by DharmaFECT transfection reagents

dharmafect editing gel  

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

dharmafect editing gel 1 

HeLa293T 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 reagents provide an improved dynamic range

dharmafect lipid volume 

DharmaFECT reagents are effective across a broader range of experimental conditions when compared to Lipofectamine® 2000 (Invitrogen). Several cell densities and lipid volumes were investigated to determine optimal transfection conditions, shown by the shaded boxes. Three cell densities of HepG2 cells were transfected with GAPD siRNA (100nM) using a range of volumes (0.05 to 1.6μL/well) of Lipofectamine 2000 and DharmaFECT 4 transfection reagents. mRNA levels (bars) were assessed by branched DNA assay (Panomics Quantigene® Reagent System) and cell viability (data points) was determined by alamarBlue® (Biosource International).

DharmaFECT Transfection Reagents provide highly efficient delivery at low siRNA concentrations

dharmafect mrna levels 

To determine transfection efficiencies at low siRNA concentrations, two genes were targeted with various amounts of SMARTpool siRNA reagent. DharmaFECT achieved > 80% silencing at all siRNA concentrations, while HiPerFect (Qiagen) only achieved this level with PPIB at 100 nM. HeLa cells were transfected with SMARTpool TM reagents targeting Cyclophilin B (PPIB) or MAPKI at concentrations of 1, 5, 25, and 100 nM. mRNA expression (bars) was determined by branched DNA assay (Panomics Quantigene® Reagent System) and cell viability (data points) was determined by alamarBlue® (Biosource International).

DharmaFECT outperforms other reagents in optimization study

dharmafect sirna transfection 

Table adapted from Borawski et al. A study by scientists at Novartis concluded that in transfection optimization in 384-well format (in preparation for screening) one of the four DharmaFECT transfection reagents outperformed all other reagents in delivery efficiency and overall cell viability in 9 of 10 cell lines. Lipid transfection reagents tested were DharmaFECT 1-4, HiPerFect® (Qiagen), TransIT-TKO® (Mirus) Lipofectamine® 2000 and Oligofectamine® (Invitrogen). J. Borawski et al., Optimization Procedure for small interfering RNA Transfection in a 384-well format. J. Bimolecular Screening,12, 546-559 (2007).