Community Research and Development Information Service - CORDIS

Development of siRNA platform for target validation

RNA interference (RNAi) is a novel target validation technology that has just come to the fore over the last 2-3 years. We have established a validation platform using small interfering RNA (siRNA), to enable us to study the phenotypic consequence of knocking out targets identified by through this collaboration, in compliance with the role for Partners 6 described in the Work Programme. Scientists at EiRx have demonstrated that by using various instructive web sites and commercial algorithms, as much as 90% of siRNA designed in-house cause greater than 60% knockdown of target mRNA.

This is also reflected in the protein levels, where western blotting of cell lysates from siRNA treated cells has confirmed that specific target protein is reduced by approximately 70 90% in most cases, 48h post treatment. EiRx also developed a transient transfection method for the delivery of siRNA into cells, with limited toxicity since the overall transfection period is 4 hours. Using a FAM (a fluorescent probe)- labelled siRNA oligonucleotide, the transfection rate for this methodology has been demonstrated to be greater than 90% across a breadth (>80) of cell lines tested to date.

As well as developing the siRNA methodology we have also developed a number of assays in order to interrogate the phenotypic consequences of targeting various cellular genes. These assays include apoptotic specific assays such as DNA fragmentation, Annexin binding and mitochondrial depolarisation (JC1), as well as clonagenic and anchorage independent assays. We have also developed siRNA compatible chemotaxis assays. We are now able to use these capabilities to examine the pro and anti-apoptotic role of novel targets in Non Small Cell Lung Cancer chemotherapy resistance.

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