Recently we want to target a gene with siRNA , but i have a critical question how can i make sure that downstream pathway of this genes will be affected . on the other hand ho can i predict bioinformatically that if the downstream pathway will be solely affected by the desired inhibited genes .
Hi Saeid,
If you want to figure out the potential downstream pathways of a target gene, you can silence this gene, and then perform microarray. However, the pathways predicted by microarray dataset are just bioinformatical analysis. You need to validate the prediction using multiple lines of experiments. For example, you can simply confirm whether the key factors downstream of this pathway are affected when the gene is silenced; accordingly, whether these changes can be reversed when you recover the gene. To go deeply, you can further figure out which step is mainly affected; for example, silencing the target gene can inhibit the phosphorylation of a certain protein, etc.
Generally speaking, there shouldn't be a pathway that is solely affected by one gene or several genes. Signaling pathways in human beings are much alike a web. They correlate to each other and form a network.
You may check the synthesis of specific proteins involved in that pathway by means of WB.
Hi
If you want to perform a bioinformatics analysis take your siRNA sequence and analyse the pathways it will target using miRNet. It is has experimentally verified data that it uses for prediction.
http://www.mirnet.ca/
Use the targets predicted to create a network in STRING or cytoscape. These are powerful tools to analyse
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