MATra Examples and Applications

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

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.

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

Transfection of Carcinoma Cell Lines 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
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). Details see application note and reference article “Renker et al. MATra- ein Trojanisches Pferd für eine zellschonende Transfektion, BIOSpektrum 2010“ (pdf, 1819 kb)
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.

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.

(Data kindly provided by Dr. Mika Ruonala, Center for Membrane Proteomics, University of Frankfurt, Germany;
ruonala@em.uni-frankfurt.de)

Suspension cells: THP-1

Transient transfection of the monocytic cell line THP1 cells

Dahl, TB and Ranheim T Institute of Internal Medicine, Oslo University Hospital Rikshospitalet.

GFP expression in THP1 cells after transient transfection with pCMV-ASC-GFP DNA (Origene). THP1 cells (1, 0.5 or 0.25 x 10⁶ cells per well in a 12 well plate) were immobilized on collagen coated plates (BD) for 24h. The cells were transfected with 1.5 µg DNA expression plasmid using MATra-A (1 µl/1 µg DNA) or Lipofectamine and MA Lipofection enhancer. GFP fluorescence was detected by flow cytometry after 22 hours.
Transfection efficiency was increased in cells transfected with MatraA compared to cells transfected with Lipofectamine and had an optimal cell density between 0.5x10⁶ and 0.25x10⁶ (table 1). Figure 1 demonstrates a representative histogram of cells seeded with a density of 0.5x10⁶ and transfected with MatraA

Figure 1. Cell sample with density of 0.5x106 and transfected with MatraA (dotted line) compared to untransfected cells (full line). Transfection efficiency 28%

Primary human chondrocytes

	Densitometric analysis of immunoblot data. Data for Jun expression were normalised to protein levels. Data show optimal expression using each transfection reagent, where ‘Con’ represents average ‘empty vector’ controls given a value of 1.

Optimisation of c-jun over-expression in primary human chondrocytes

Method. Primary human articular chondrocytes isolated from cartilage of patients undergoing total knee replacement were cultured for approximately 10 d before trypsinisation and re-seeding into 6-well dishes at approx. 70% confluence. Cells were transfected with pCMV2 harbouring human c-jun or empty vector using either FuGENE HD or MATra-A transfection reagents, using protocols and a range of DNA:reagent ratios as suggested by manufacturers. After 24 h cells were lysed and 10 μg lysates subjected to SDS-PAGE and immunoblotting using α-Jun antibody (Santa Cruz).

Transfection of insect cells

Transfection of HUVEC with siRNA

80% down-regulation of the protein!

Human umbilical vein endothelial cells (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
Figure above: Zyxin knock down by Magnet Assisted Transfection (MATra). Immunofluorescence analysis of HUVEC. Representative confocal immunofluorescence images of zyxin (Cy3/red) in non-transfected human cultured endothelial cells (HUVEC; left picture) and HUVEC after Magnet Assisted Transfection (as described in the text; right picture). The cells were counter-stained with the focal adhesion protein paxillin (Cy2/green) and the DNA-binding nuclear dye DAPI (blue). Paxillin co-localizes with zyxin exclusively in the focal adhesions (yellow) but not in the stress fibers or in the nucleus.

Choriocarcinoma cells: BeWo

BeWo cells were efficiently transfected with an expression vector coding for green fluorescent protein (GFP) using magnet assisted transfection.
Therefore, BeWo cells were seeded 24 h before transfection in a 12well plate at a density of 180 000 cells/well. 1µg, 2µg and 3µg of plasmid respectively were incubated in 200µl OptiMEM^® I Reduced Serum Medium with Lipofectamine in a ratio of 1:3 for15 minutes. MA Lipofection Enhancer reagent was added in a ratio 1: 1.2 with an additional incubation time of 15 minutes. The plate was placed on the MATra Magnet Plate for 15 minutes at room temperature to allow beads to penetrate the target cells. GFP fluorescence was detected by flow cytometry after 36 hours.
 (Data kindly provided by Andrea Milenkovic, Human Genetics Regensburg, Germany)

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Fig.: FACs analysis of GFP transfected BeWo cells  not transfected  1µg Plasmid + 3µl Lipofectamine + 1.2µl MA Lipofection Enhancer  2µg Plasmid + 6µl Lipofectamine + 2.4µl MA Lipofection Enhancer  3µg Plasmid + 9µl Lipofectamine + 3.6µl MA Lipofection Enhancer