MATra™ Data, Images and Testimonials

MATraTM Images, Data and Testimonials for siRNA, DNA and ODN Applications

Small inhibitory RNA oligonucleotides (siRNAs) allow transient inhibition of gene expression in vitro and in vivo.

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

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

 

 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/Neuromics 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

Example 1

 

Embryonic cortical neurons were transfected with human NCAM. After transfection membrane-localized NCAM (not endocytosed) was detected using a Cy3-coupled secondary antibody (red). Afterwards, the internalised, endocytosed NCAM was stained by a Cy2-coupled secondary antibody (green, see arrows) in the cell soma (left) and in axonal vesicles (right).

 Example 2

 

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

Cerebellar granular cells from CD1 mice
 

Cultured cerebellar granular cells from CD1 mice were transfected by below 4 constructs (A-D) using MATra-A.
(A) MyrPalm-mCFP, cyan (provided by Dr. R. Tsien, UCLA)
(B) Actin-DsRed, red
(C) Flotillin-2-mVenus, yellow (B and C provided by Dr. R. Tikkanen, University of Giessen)
(D) Battenin-myc, detected by using GAM-Alexa647, dark green
(E) Surface: Crop of the whole image with 3D surface rendered fluorescence signals overlayed on phase contrast image.
(F) PhaCo: Phase contrast image

Primary hippocampal neurons (E14)
 
  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, Center for Membrane Proteomics, University of Frankfurt.

MATra – best transfection practices for Cancer Research

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

Transfection of APP into neuroblastoma

 
 

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 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

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).

 

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’.

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).

Human endometrial stromal cells transfected with siRNA
 
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)