Neuroscience-Best Transfection Practices

I have multiple posting of customer success in transfecting siRNA and plasmids into neurons using Neuromics' i-Fect^TM and pn-Fect^TM...related publications.

Here I post excellent data, images and testimonials provided by researchers using our Magnetic Assisted Transfection (MATraTM). Our goal is to continue our journey towards having the best practices for gene expression analysis studies in the CNS.

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. 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 pPrimary 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 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 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  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. 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.

Using MATRa for siRNA Transfection of Carcinoma Cell Lines

MATRa^TM -Magnet Assisted Transfection is an easy-to-handle, very fast and highly efficient technology to transfect cells in culture with siRNA. Multiple successes with the system includes Carcinoma Cell Lines.

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