- If mRNA is degraded there is no gene expression ("Gene silencing").
siRNA Transfection for Interference Studies
siRNAs are small interfering/inhibitory sequence-specific RNA molecules that are able to bind to a complementary sequence of a target mRNA. This results in blocking translation and initiating degradation of that target mRNA, thus preventing the synthesis of the corresponding protein of interest. This is termed RNA interference or gene-silencing and is used to elucidate gene functions. Small inhibitory RNA oligonucleotides allow transient inhibition of gene expression in vitro and in vivo.
MATra is an excellent tool to transfect siRNA into many different cell lines, such as carcinoma cell lines (see examples below).
MATra-A Reagent has successfully been used to deliver siRNA in a variety of cell types. However, for optimized siRNA transfection and prolonged gene silencing PromoKine provides the specifically developed MATra-siRNA Reagent.
Examples & Applications
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 for IBA GmbH by Dr. Birgit Gellersen, Endokrinologikum Hamburg, Hamburg, Germany)
- Left: Fluorescent
- Right: Phase contrast
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/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 /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 for IBA GmbH 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.
Note: Cell Transfection (e.g. transfection of primary human cells) is also topic of a training course of our PromoCell Academy! >More Information