Effective silencing of targeted gene using low siRNA concentrations.
Transfection of a wide variety of cell types.
Compatible with serum and antibiotics.
Applications
siRNA transfection.
siRNA-DNA co-transfection.
Description
TurboFect™ siRNA Transfection Reagent is a sterile solution of a cationic polymer in water. The polymer forms compact, stable, positively charged complexes with siRNA and facilitates efficient siRNA delivery into the cytoplasm of cells. The reagent delivers siRNA into a variety of cell types, including primary and suspension cells and allows the efficient gene silencing using low siRNA concentrations. TurboFect™ siRNA Transfection Reagent is also ideal for siRNA-DNA co-transfection. Transfection is highly efficient in the presence or absence of serum and antibiotics.
HeLa human cervix adenocarcinoma cells HR5-CL11 human cervix adenocarcinoma cells, HeLa derivate CHO chinese hamster ovary cells NIH3T3 mouse embryo fibroblasts
Figure 1. Fluorescent image of HeLa cells transfected with Cy3-labeled siRNA (red) using TurboFect™ siRNA Transfection Reagent. The cells were fixed 2 hours after transfection, nuclei (blue) stained with DAPI, endosomes (green) labeled with anti-transferrin receptor antibodies. Confocal microscope image, cross-section through the nucleus.
Figure 2. Efficient GFP knockdown after siRNA transfection using Fermentas TurboFect™ siRNA Transfection Reagent. NIH3T3 cells constitutively expressing the gfp gene were transfected with GFP targeting siRNA and a non-targeting siRNA control. GFP expression was measured by RT-qPCR 48 hours after transfection. GAPDH was used as an endogenous gene control.
Figure 3. Inhibition of GFP in cells transfected with GFP-targeting siRNA using Fermentas TurboFect™ siRNA Transfection Reagent and reagents from other vendors. NIH3T3 cells constitutively expressing the gfp gene were transfected with GFP targeting siRNA and a non-targeting siRNA control according to each manufacturers recommended protocol. 48 hours after transfection, the amount of mGFP was measured using RT-qPCR. GFP expression was normalized to the expression of the c-myc gene. Specific and non-specific siRNA transfection data was used to calculate relative GFP mRNA expression.
Figure 4. Inhibition of GFP in cells transfected with GFP-targeting siRNA using Fermentas TurboFect™ siRNA Transfection Reagent and reagents from other vendors. NIH3T3 cells constitutively expressing the gfp gene were transfected with 3 pmol (5 nM final siRNA concentration) of GFP-targeting siRNA and a non-targeting siRNA control. 48 hours after transfection, GFP fluorescence in cells transfected with GFP targeting siRNA was measured relative to cells transfected with non-targeting siRNA. The half-life of GFP is 26 hours
siRNA Transfection using TurboFect™ siRNA Transfection Reagent
Reagents to be Supplied by the User: 0.15 M NaCl, serum-free DMEM, RPMI or other serum-free medium. The protocol below is provided for adherent and suspension cells in a 24-well plate using TurboFect™ siRNA Transfection Reagent (#R1401). Quantities and volumes should be scaled-up according to the number of cells/wells to be transfected (see Table 1). Subsequent optimization of the quantities of siRNA and transfection reagent may further increase the transfection efficiency and result in more efficient gene silencing. Protocol
Seed ~5x104 adherent cells or ~1x105 suspension cells per well in 0.5 ml of growth medium 24 hours prior to transfection.
Note
The recommended confluency for adherent and suspension cells on the day of transfection is 70-90%.
Dilute 3 pmol of siRNA in 100 μl of 0.15 M NaCl or other serum-free medium for a final siRNA concentration of 5 nM in the cell culture.
Add 1 μl of TurboFect™ to the diluted siRNA and mix by pipetting.
Incubate 15-20 minutes at room temperature.
Note
The TurboFect™/siRNA complexes should be prepared immediately prior to transfection.
Add 100 μl of the TurboFect™/siRNA mixture drop-wise to each well.
Gently rock the plate to achieve an even distribution of the complexes.
Incubate at 37°C in a CO2 incubator.
Assay gene suppression 24-72 hours later.
Table. Recommended number of cells to plate 24 hours prior to transfection with TurboFect™ siRNA Transfection Reagent.
Tissue culture vessel
Growth area, cm2/well
Media, ml
Adherent (suspension) cells to seed the day before transfection*
96-well plate
0.3
0.1
0.5-1.20 x 104 (2.0 x 104)
48-well plate
0.7
0.25
1.0-3.0 x 104 (5.0 x 104)
24-well plate
2.0
0.5
2.0-6.0 x 104 (1.0 x 105)
12-well plate
4.0
1.0
0.4-1.2 x 105 (2.0 x 105)
6-well plate
9.5
2.0
0.8-2.4 x 105 (4.0 x 105)
60 mm plate
20.0
3.0
2.0-6.3 x 105 (1.0 x 106)
Note
* These numbers were determined using NIH3T3 GFP expressing cell line. Actual value depends on the cell type.
- Worldwide
- to print
- Store at 4°C
Figure 1. Fluorescent image of HeLa cells transfected with Cy3-labeled siRNA (red) using TurboFect™ siRNA Transfection Reagent.
Figure 2. Efficient GFP knockdown after siRNA transfection using Fermentas TurboFect™ siRNA Transfection Reagent.
Figure 3. Inhibition of GFP in cells transfected with GFP-targeting siRNA using Fermentas TurboFect™ siRNA Transfection Reagent and reagents from other vendors.
Figure 4. Inhibition of GFP in cells transfected with GFP-targeting siRNA using Fermentas TurboFect™ siRNA Transfection Reagent and reagents from other vendors.
