Tools and technology:
By Brona McVittie
“We are building an overall picture, a bit like a railway map,” explains Michael Boutros. “We know where the tracks go through, but there are a lot of blanks to fill in, like which factors affect the whole system. In terms of methodology, we take lots of different snapshots of the system in a rapid manner. So we hope to identify critical intervention points simultaneously rather than one by one. We call this high-throughput screening.”
Custom designed siRNAs are applied to cells in tiny wells on a microplate. Microplates contain hundreds of wells and so many different siRNAs can be applied across a plate to the same cell types. ‘Knock down’ experiments can thus be run simultaneously for many genes. The activity of a particular pathway can be estimated by measuring the expression of certain target genes.
Now that it is possible to run thousands of experiments simultaneously, new techniques are required to organise and analyse the huge amount of data generated. The results are organised into a database structure. A variety of mathematical algorithms may then be applied to reveal meaning from the data. The development of computational and statistical techniques to this end encompasses the field of bioinformatics.
The CancerPathways network is focused on making high-throughput RNAi screening more feasible. As Michael explains, “We’re using RNAi as a tool to switch off genes and identify components of signaling pathways.” High-throughput screening allows a researcher to quickly conduct millions of simultaneous experiments. “Now that we know the sequences of genes in the genome, we can attack every single one using RNAi reagents that we design from scratch.”
Although high-throughput RNAi can quickly uncover pathways components, their function(s) in living animals requires further investigation. So the CancerPathways team is generating transgenic fly strains each capable of expressing double-stranded RNA designed to knock down one gene in the genome. This library of transgenic RNAi fly lines allows scientists study the function of genes in living tissues.