IBA : MATra

siRNA
Neurosciences
Insect cells
HUVEC
Mouse fibrosarcoma
FaDu carcinoma
Hepatocellular carcinoma
Primary hepatocytes
VSa13
Comparison of transfection rates of various cell lines

For further examples see the MATra references.

siRNA

Small inhibitory RNA oligonucleotides (siRNAs) allow transient inhibition of gene expression in vitro and in vivo.
MATra is an excellent tool to transfect siRNA into different cell lines, such as carcinoma cell lines. MATra-si Reagent is optimized for siRNA applications
Being a specialist for RNA synthesis we are also offering siRNA for gene silencing studies.
Short RNA duplexes are offered ready-to-use, i.e. purified, deprotected and annealed. A negative control sense and antisense RNA are included. There are no additional charges for the number of base pairs. siRNA is also available labeled with fluorescent dyes or biotin.

Magnet Assisted Transfection of primary rat hepatocytes

See also transfection of hepatocytes with plasmid DNA.
Primary hepatocytes are of great scientific interest and are commonly used as a model system to study mechanisms of cell growth and differentiation, bio-transformation, drug pharmacokinetics and toxicity of drugs. However, most methods fail to efficiently transfect primary hepatocytes.

Magnet Assisted Transfection technology enables:

Transfection of slowly dividing cells such as hepatocytes
A non-viral transfection method for cells, that are difficult to transfect
High transfection efficiencies using DNA, ODN* and siRNA

Knockdown of inducible nitric oxide synthase (iNOS) mRNA by sense ODN and siRNA

Mikio NISHIZAWA, Prof., Dr.
College of Life Sciences, Ritsumeikan University. Kusatsu, Shiga, Japan.
e-Mail: nishizaw@sk.ritsumei.ac.jp

Using MATra-A Reagents, our group has successfully introduced plasmid DNA and oligodeoxyribonucleotides (ODNs) into primary cultured rat hepatocytes and murine RAW264 macrophage cells [1–7]. Sense ODNs (i.e., single-stranded DNA) to iNOS mRNA inhibit the interaction between the iNOS mRNA and antisense transcript to knockdown iNOS mRNA [8]. To compare degradation efficacy of the sense ODN and siRNA (i.e., double-stranded RNA), we transfected rat hepatocytes and estimated iNOS mRNA levels by reverse transcription and real-time PCR. As shown in Fig. 1, the sense ODN S4 corresponding to iNOS gene exon 27 [8] reduced the level of iNOS mRNA to 63.9% to that of mock transfection. We designed siRNAs (siE26 and siE27) corresponding to iNOS gene exon 26 and 27, respectively. When they were introduced, iNOS mRNA was reduced to the levels which were comparable to that when S4 was introduced. Considering that the transfection efficiency is about 50 %, it demonstrated that both siRNAs and the sense single-stranded ODN to iNOS mRNA are successfully introduced to rat hepatocytes and effectively degrade iNOS mRNA.

"MATra is a powerful tool to transfect difficult cells, such as primary cultured rat hepatocytes, with ALL the types of DNA/RNA including plasmid DNA, ODN, and siRNA."

Prof. Dr. Nishizawa, Ritsumeikan University, Japan

References
1) Tanaka H, et al. J Hepatol. 48:289–299 (2008).
2) Yoshida H, et al. Nitric Oxide. 18:105–112 (2008).
3) Tokuhara K, et al. Nitric Oxide. 18:28–36 (2008).
4) Habara K, et al. Nitric Oxide. 18:19–27 (2008).
5) Matsui K, et al. JPEN J Parenter Enteral Nutr. 31:373–380 (2007).
6) Ozaki T, et al. JPEN J Parenter Enteral Nutr. 31:366–371 (2007).
7) Yoshida H, et al. Shock. 30:734–739 (2008).
8) Matsui K, et al. Hepatology. 47:686–697 (2008).

Both a sense ODN and siRNAs effectively degrade iNOS mRNA. Rat hepatocytes (1.2 x 106 cells/well) were transfected with 1 mg ODNs (S4 and Scr4) or siRNAs (siE26, siE27, and siScr) using 1 mL MATra-A Reagent. Next day, total RNA was extracted from IL-1b-treated hepatocytes and subjected to real-time PCR of iNOS and EF-1a mRNA (n=3), as previously described [8]. Expression levels of iNOS mRNA were normalized by those of EF-1a mRNA. (–), mock transfection. S4, 5’-GTGTATAATTCCTTGATGAA-3’; Scr4, a scrambled control of S4, 5’-GATTGTTACTTAGAGACTAT-3’; siE26, 5’-CCAGCUCAAGAGCCAGAAAdTdT-3’ (sense strand is shown); siE27, 5’-CCGUGUAUAAUUCCUUGAUdTdT-3’; and siScr, a scramble control of siE27, 5’-GCUUGCUAUGUACACUUAUdTdT-3’.

Human endometrial stromal cells transfected with siRNA


Fluorescent	Phase contrast

Primary cultures of human endometrial stromal cells were plated in a 96 well plate at a density of 13,000 cells/well. Twenty-four hours later medium was changed. Fluorescein-siRNA was diluted in OptiMEM^® I Reduced Serum Medium (GIBCO) to 0.9 µg/108 µl. MATra-A reagent (0.45 µl) was added to obtain a ratio of 2:1 (siRNA:MATra-A). After 20 minutes incubation at room temperature, 15 µl of the mixture (corresponding to 125 ng siRNA) were added per well. The culture plate was placed on a 96 Magnet Plate for 15 minutes at 37°C. Cells were incubated for 20 hours at 37°C, medium was changed before microphotographs were taken. Virtually all cells had taken up the fluorescent siRNA. (Data kindly provided by Dr. Birgit Gellersen, Endokrinologikum Hamburg, Hamburg, Germany)

Transfection of Carcinoma Cell Lines with siRNA

Efficient transient transfection of siRNA in head and neck cancer cells. The cell line ANT-1 was transiently transfected with MATra-A (1 µl/1 µg DNA) in a 6 well format (5 x 10⁵ cells/cavity) with siRNA against protein 1 (100 nM). After 24 hours total RNA was isolated and expression of protein 1-specific mRNA determined by RT-PCR (upper lane). SiRNA 13 are three different oligonucleotide sequences. Control for consistent loading and cDNA quality: expression of ubiquitary GAPDH mRNA (lower lane).
Protein 2 expression in head and neck cancer cells GHD-1. GHD-1 cells (5 x 10⁵ cells/cavity of a 6 well plate) were transiently transfected with two different siRNAs against protein 2. Expression of protein 2 was detected with specific antibodies in an immunoblot 72 hours after transfection with MATra-A (1 µl / 1 µg DNA). As control ubiquitary β-actin was detected as well.
Treating the carcinoma cells with specific siRNA caused a clear inhibition of protein 1/protein 2 expression which indicates high transfection efficiencies.
(Data kindly provided by Rauch, Schaffrik, Ahlemann and Gires, LMU Munich and GSF, Munich, Germany)

"After having tested MATra in a variety of experimental set ups we can summarize the following advantages:

High transfection efficiency
Easier to handle
High reproducibility
Serum compatibility
Low sensibility against cell confluence"

Dr. Oliver Gires, LMU Munich, Germany

See also Transfection of HUVEC with siRNA

MATra – best transfection practices for Neurosciences

Neurosciences are a vast and expanding field of research focussing on highly sophisticated and enthralling questions. With Magnet Assisted Transfection IBA offers a very gentle and potent tool for the transfection of many kinds of neuronal cells. Magnet Assisted Transfection is the ideal solution to overcome problems related to the study of complex and easily interrupted systems.

Transfection of primary cortical neurons

Primary cortical neurons from mice embryonic day 15.5 (E15.5) were grown on poly-L-lysine coated coverslips at a density of 800.000 cells/well in a 24-well plate. The neurons were transfected after 1 day in vitro (DIV 1) with pCX-EGFP-N1 plasmid. Transfection was carried out as recommended by the manufacturer (0.6 µg DNA, 0.6 µL Matra-A reagent). Cells were fixed 24 h later (DIV 2) and GFP fluorescence was visualized using a confocal laser scanning microscope.

"With Matra we achieved a higher transfection efficiency than with different liposomal transfection methods and no toxicity to the cells was observed."

Dr. Simone Diestel, Institute of Animal Science, University Bonn, Germany

Transfection of APP into neuroblastoma using MATra

Cells maintain their endogenous expression pattern and stay unaffected from transfection related influences:
B103 neuroblastoma cells were plated at 105 cells/well in Dulbecco’s modified Eagle’s medium + 10% fetal calf serum on poly-L-ornithine-coated glass coverslips in 24-well plates (Corning Life Sciences, Lowell, MA) and transfected using 0.2–0.8 μg of plasmid DNA per well and MATra-A beads on a 24 Magnet Bar Plate. The medium was changed 1–2 hrs after transfection, and expression was allowed to proceed for a further 16–24 hrs.
Figure: Investigation of APP dimerization using APP-GFP. A, confocal image of a B103 cell expressing APP-GFP. B–G, wide-field images of B103 cells expressing APP-GFP alone (B–D) or in combination with APP-mCherry (E–G). B and E, GFP channel. C and F, mCherry channel. D and G, GFP lifetime. Scale bars: 10 μm. H, histograms of FRET efficiencies in different experimental conditions. PDF, probability density function.
Expression levels were high enough to aquire fluorescence lifetime images (Fig. B-G), which permitted calculating the levels of interaction between APP-GFP molecules in the cell (Fig. H)
Data kindly provided by Dr. Matthias Gralle, Max-Planck-Institute for Evolutionary Anthropology, Leipzig, Germany, Gralle et al. (2009) J Biol Chem 284, 15016-25.

"Several liposomal methods were tried out, but the transfection efficiency was low, and the transfected cells were rounded and visibly unhealthy. With MATra, the expression pattern of APP-GFP was indistinguishable from the known expression pattern of endogenous APP, and the cells maintained the typical elongated morphology with protrusions", said Dr. Gralle at MPI, Leipzig, Germany.

Neurons transfected with eGFP plasmid

Primary hippocampal neurons (E14) were grown on 15 mm glass coverslips on a 12 well at density of 150.000/cm². The neurons were transfected 4 d.i.v. with pSyn-eGFP using 25 µl MATra complex per well (prepared by adding a MATra-A Reagent-DNA complex mixture (2.8 µg cDNA; 2.8 µl beads) into 175 µl neuronal medium without serum). The cells were fixed 6 d.i.v. with 4% PFA and imaged.
"With MATra we can transfect and modulate the expression levels of exogenous proteins in highly sensitive primary neurons without any toxicity. Once optimized, double and even triple transfections with different DNA ratios are easily achieved" said Dr. Mika Ruonala, ENI, Göttingen.
(Data kindly provided by Dr. Mika Ruonala, Center for Membrane Proteomics, University of Frankfurt, Germany;
ruonala@em.uni-frankfurt.de)

Transfection of insect cells

"We have used MATra-A as an alternative to electroporation for Sf9 insect cell transfection with Baculovirus and have been excited about the performance: The MATra approach is extremely gentle and does not cause the cell death we often observe with the harsh electroporation procedures. Therefore, we now routinely use the MATra system for insect cell transfection."

Dr. Rudolf Hauptmann, Boehringer Ingelheim RCV GmbH & Co KG, Austria

For further insect cells (Mamestra brassicae) refer also to our table of "Successfully Transfected Cells"

Transfection of HUVEC with siRNA

80% down-regulation of the protein!

HUVEC were efficiently transfected with short interfering RNA (siRNA) using magnet assisted transfection. Briefly, 3 µg of siRNA were diluted in OPTIMEM I medium to give a final volume of 200 μl for each well to be transfected (final concentration approximately 30 nM). For the formation of the transfection complex, 3 μl of MATra-si reagent were added to the diluted siRNA, carefully mixed and allowed to incubate at room temperature for 25 minutes. HUVEC were once washed in OPTIMEM I, the medium was discarded and then fresh OPTIMEM I (2 ml per well) was added to the cells. The siRNA/magnetic beads were then overlaid drop wise onto the cells (200 μl/well). Cells were incubated with the transfection complex on a custom made magnetic plate which was specifically designed to fit the wells of Flexcell plates (Universal Magnet Plate, IBA) for 15 minutes in the cell incubator to allow beads to penetrate the target cells. Thereafter, to induce rapid zyxin turn over, cells were shortly exposed to cyclic stretch (30 minutes, 0.5 Hz, 10 % elongation¹). After this manoeuvre, the medium was changed to prevent cytotoxic effects from the transfection reagent, and the cells were kept further under their normal culture conditions. Gene silencing was optimal 72 hours after transfection.

Other gene products knocked down with similar efficiencies: Ku70/Ku80, PARP1, hnRNP U.

zyxin ~ 80kDa

ß-actin as loading control

Control cells

2 Hs_Zyxin_1 HP siRNA

"We have tried to efficiently transfect siRNA into Huvec for month with only minor success (up to 8% efficiency). With Magnet Assisted Transfection using the MATra reagent we finally achieved a knock down of 80% – definitely a break through."

PD Dr. Marco Cattaruzza, Institute for Physiology and Pathophysiology, Heidelberg University, Germany

¹Zyxin is a very stable protein, thus, an efficient knock down is difficult to achieve. Under conditions of mechanical strain, the protein translocates to the nucleus where it regulates gene expression. In the nucleus, however, zyxin is by far less stable, i.e., it seems to undergo accelerated export and degradation. Therefore, stretching the cells is a zyxin-specific manoeuvre aiming at rapidly decreasing the pre-transfection levels of zyxin. In our hands, when attempting to knock down stable proteins, it is a good idea to try to accelerate their turn over, e.g., by (shortly) activating cellular pathways involving the protein in question.

Mouse fibrosarcoma cells transfected with GFP plasmid


Phase contrast	GFP fluorescence	Overlay

Magnet Assisted Transfection (MATra) of L929 fibrosarcoma cells. 1 x 10⁵ L929 cells were seeded on poly-L-lysine coated glass coverslips and allowed to grow for 24 h. Subsequently, the cells were transfected with 1 µg of an expression vector coding for green fluorescent protein (GFP) as described in the standard protocol for MATra. 48 h after transfection, the cells were fixed with 4% (w/v) paraformaldehyde and expression of GFP was visualized by confocal laser scanning microscopy.
Transfection efficiency was 60 - 80% (see overlay).
(Data kindly provided by Dr. Lutz Thon and Dr. Dieter Adam, Institut für Immunologie, Universitätsklinikum Schleswig-Holstein Campus Kiel, Kiel, Germany)

Transient transfection of stable carcinoma cells with GFP plasmid

GFP expression in FaDu head and neck cancer cells after transient transfection with pGFP plasmid DNA. FaDu cells (5 x 10⁵ cells per cavity of a 6 well plate) were transfected with 1.0 µg (B) or 1.5 µg (C) pGFP expression plasmid using MATra-A (1 µl/1 µg DNA). Control: 1.0 µg empty vector, transfected under same conditions (A). GFP fluorescence was detected by flow cytometry after 48 hours.
FaDu cells are typically transfected with standard lipofection reagents with an efficiency of about 10% (1 µg GFP in 5 x 10⁵ cells in 6 wells).
With MATra expression of GFP was detected in 52.7% (1.0 µg) and 82.55% (1.5 µg) of the cells.
"With MATra we have been able to increase the transfection efficiency to rates as high as 80% at 48 h following treatment" stated Olivier Gires from the LMU Munich. "All cell lines tested showed an increased transfection rate with MATra-A in comparison to lipofection or electroporation protocols."
(Data kindly provided by Rauch, Schaffrik, Ahlemann and Gires, LMU Munich and GSF, Munich, Germany)

With MATra transfection efficiency has been
increased 8x compared to lipofection.

Human hepatocellular carcinoma cells transfected with GFP plasmid


MATra-A	Competitive lipofection reagent

Magnet Assisted Transfection of hepatocellular carcinoma cells (Hep G2) was compared to lipofection. Transfections with pCG-IRES-GFP (own construct) were carried out in 96 well plates according to standard protocols without medium change. Cells were fixed with 2% PFA 24 hours post transfection for fluorescence microscopy. Confluency ~ 80 - 90%.
MATra shows much higher transfection efficiency than competitive lipofection reagent.
(Data kindly provided by Michael Schindler, University Ulm, Microbiology and Virology, Ulm, Germany)

Magnet Assisted Transfection (MATra) of primary cultured rat hepatocytes

"For primary cultured rat hepatocytes MATra has been the most efficient transfection method we have tried so far, and it is much more cost-effective than the common lipofection reagents on the market."
Prof. Dr. Okumura, Kansai Medical University, Osaka, Japan

Hepatocytes prepared from liver were seeded on 3.5 cm diameter dishes (3-5x10⁵cells/dish) and allowed to grow overnight. The cells were transfected with pCMV-LacZ, a CMV enhancer/promoter-driven β-galactosidase plasmid, as described in the standard protocols for MATra. The cells were fixed with 1% glutaraldehyde and stained in 2 mg/ml X-Gal solution. β-galactosidase-expressing blue cells were examined by microscopy.
With MATra-A a minimum of 5% cells expressed β-galactosidase, which was several fold better than with lipofection.
(Data kindly provided by Prof. Mikio Nishizawa and Tadayoshi Okumura, Dept. Biomedica Sciences, Ritsumeikan University, Kusatsu, shiga, Japan).

See also transfection of hepatocytes with siRNA

Magnet Assisted Lipofection of fish bone-derived VSa13 cells

	Luciferase activity	Conditions
A	1	3 µl of Lipofection Reagent "F6"/500 ng GAL-LUC and 50 ng CMV-GAL
B	7.59	like A, plus 1 µl of MA Lipofection Enhancer
C	64.75	like B, but 2500 ng GAL-LUC and 250 ng CMV-GAL

The fish bone-derived VSa13 cells were cultured in D-MEM supplemented with 10% FBS. Magnet Assisted Lipofection was performed in cultures at 60 - 80% confluence grown in 12 well plates and in absence of FBS. CMV-GAL and a GAL-LUC constructions were co-transfected using MA Lipofection Enhancer combined with Lipofection reagent "F6". (Alternatively, IBA's lipofection reagent IBAfect can be used!)
Especially for cells difficult to transfect, it is important to titrate the optimal DNA concentration to obtain highest transfection efficiencies. In VSal3 fish bone-derived cells, multiplying DNA quantity by 5 resulted in about 8.5-fold increase in transfection efficiency.
(Data kindly provided by Vincent Laizé, Universidade do Algarve, Faro, Portugal)

Comparison of transfection rates of various cell lines

HeLa
CHO-K1
	Standard transfection method	Magnet Assisted Transfection	For indicated cells the following methods were tested. 1: Calcium phosphate vs Magnet Assisted Lipofection, 2-4 Lipofection vs Magnet Assisted Lipofection, 5: Lipofection vs Magnet Assisted Transfection. Data kindly provided by industrial IBA customer.
	Data kindly provided by Dr. Günther Keil, Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, Island Riems, Germany.



		MATra Examples and Applications

MATra Examples and Applications