Keywords: siRNA High-throughput screening, low siRNA concentration,
high transfection efficiency, no toxicity, gene silencing.

Synvolux Therapeutics siRNA transfection reagent SAINT-RED permits the rapid and
efficient delivery of siRNA into a variety of mammalian cell lines. Here, the application
of SAINT-RED in siRNA high-throughput screening is demonstrated.

To unravel the detailed function of all genes in the human genome is one of the major challenges,
now that the complete human genome has been sequenced. RNA interference is a natural way by
which living cells silence individual genes. This method is also used by researchers to
investigate the function of specific genes. First, the mRNA encoding the protein has to be
identified. Then, the sequence of this mRNA is used as input for sophisticated algorithms,
which predict the most potent siRNAs. Sometimes, three 21-mer siRNA oligonucleotides are
chemically synthesized to obtain maximal silencing. The siRNAs then have to be delivered to the
living cells to study the effect on protein expression. One of the means to deliver siRNA to a
living cell is to wrap it in a lipid mixture specially designed by Synvolux Therapeutics
to chemically stabilize siRNA molecules. This lipid mixture, called SAINT-RED also enhances
the intracellular release of siRNA, thus minimizing the amount of siRNA needed.

In high-throughput siRNA screening 96 or 384 well plates are used in which the siRNA transfection
is done. First, the transfection complex of siRNA and SAINT-RED is placed in the well, and then the
cells are added on top. This is called “reverse transfection”, as opposed to “forward transfection”
in which the cells are first seeded, followed by addition of the transfection complex the next day. Of
both methods reverse transfection is the easiest and fastest. After 24 to 96 hours the cellular
phenotypes can be analysed using confocal high-content screening systems.

When the question is which genes are involved in bringing about a change in a cellular phenotype, ideally one wants to screen all known genes for involvement in this change. At this moment it is feasible to screen all known 23,000 human genes, using 3 siRNAs per gene, by means of a high-throughput system within a week or so. For academic institutions and big pharmaceutical companies it has been worthwhile to set up this kind of high-content high-throughput screening facilities. At one of this facilities, the Max Planck Institute of Molecular Cell Biology and Genetics, SAINT-RED has been tested for application in high-throughput siRNA screening in 96 well plates on HeLa cells. In this test, a positive versus a negative control siRNA have been tested to assess efficiency, cytotoxicity and off-target effects due to too efficient delivery resulting in an intracellular excess of siRNA clogging the RNA interference machinery. It also has been shown that both the forward and reverse transfection protocol yield similar results. Eberhard Krausz, Head of the High-throughput Technology Development Studio at the Max Planck Instute says: “For 10nM and 30nM siRNA concentration on the cells, there is a great window between efficiency and any unwanted unspecific effects”. No toxic effects were seen in the range from 0.1 to 0.45µL SAINT-RED per well (figure 1).

References:

Krausz E., High-content siRNA screening, Mol Biosyst. 2007 Apr;3(4):232-40.

Eberhard Krausz
Head, HT-Technology Development Studio (TDS)
Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG),
Pfotenhauerstrasse 108, D-01307 Dresden, Germany.
WWW: http://tds.mpi-cbg.de/webtds/