RNA Interference in Mammalian Cells Using low siRNA Concentrations
Poster Feb 01, 2007
Jörg Dennig, Silvia Magyar, Anja Grewe, Cornelia Schmidt, Peter Hahn, Dong Liang, Subu Yerramilli, Eric Lader, Wolfgang Bielke, and Jie Kang
The use of short interfering RNA (siRNA) for knockdown of gene expression has become a powerful tool in molecular and cell biology. Some applications require the use of low siRNA concentrations (less than 5 nM), for example, to decrease the possibility of non-specific effects.
We have developed a transfection reagent, HiPerFect Transfection Reagent, which allows efficient gene knockdown with siRNA concentrations from 1 nM–10 nM, depending on the cell type and siRNA used. HiPerFect Transfection Reagent has been tested and validated for many cell types, including primary cells. Effective knockdown in primary cells demonstrates that HiPerFect Transfection Reagent ensures low cytotoxicity levels.
A Fast-Forward siRNA Transfection Protocol has been developed for rapid transfection with HiPerFect Transfection Reagent. This protocol allows cell seeding and transfection on the same day.
A reverse transfection protocol has been developed that is ideal for use in high-throughput applications. In reverse transfection, siRNA is spotted into wells, followed by addition of HiPerFect Reagent. After complex formation, cells are added to the wells.
Despite the developments in conventional PCR, the complexity of multiplex Real Time PCR is still limited due to the lack of sufficient detection channels. To achieve high-end multiplexing capacity on standard Real Time PCR machines, Anapa Biotech has developed the MeltPlex® technology (see box on right).READ MORE
Genome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes. The majority of these are located in the intergenic or intragenic regions suggesting that the implicated variants may alter chromatin conformation. This, in turn, is likely to influence the expression of nearby or more remotely located genes to alter beta cell function. At present, however, detailed molecular and functional analyses are still lacking for most of these variants. We recently analysed one of these loci and mapped five causal variants in an islet-specific enhancer cluster within the STARD10 gene locus. Here, we aimed to understand how these causal variants influence b-cell function by alteration of the chromatin structure of enhancer clusterREAD MORE
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