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Transfection Reagents

TurboFect™ siRNA Transfection Reagent

Product information

Features

• Highly efficient siRNA transfection.
• siRNA-DNA co-transfection.
• 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.

Storage

Store 4°C.

Successfully Transfected Cells

Permanently growing cell lines

CHO chinese hamster ovary cells
HeLa human cervix adenocarcinoma cells
HR5-CL11 human cervix adenocarcinoma cells, HeLa derivate
NIH3T3 mouse embryo fibroblasts

Primary cell cultures

HLF human lung 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

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

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

1. 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%.

2. 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.
3. Add 1 μl of TurboFect™ to the diluted siRNA and mix by pipetting.
4. Incubate 15-20 minutes at room temperature.

Note

The TurboFect™/siRNA complexes should be prepared immediately prior to transfection.

5. Add 100 μl of the TurboFect™/siRNA mixture drop-wise to each well.
6. Gently rock the plate to achieve an even distribution of the complexes.
7. Incubate at 37°C in a CO₂ incubator.
8. 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.