How to validate genes identified computationally

Question

[MAPK]

I am not much familiar with the molecular techniques to functionally validate the genes that are identified to be associated with certain diseases. I have identified some somatic mutations in cancer and would like to know all available methods that I can adopt to validate these mutations. Only thing I can think of is performing Sanger sequencing to check if the mutations are real, and RT-PCR to see if this gene is expressed in the cancer I am studying. What other methods (from simple to more sophisticated) are there to validate the roles of these genes/mutations fulfilling publication criteria?

[Louis Kok]

If you have identified the causal gene, you can probably design the primers to flank the regions of the gene. Make sure to check for primer specificity to avoid unwanted regions to be amplified from the genome. After that, the amplified PCR products can be validated using targeted sequencing by Sanger, or NGS if affordable. For RNA samples, RT-PCR is a good idea but you will also need control samples (e.g. gene sequence of healthy individual) to to compare the expression pattern. Targeted RNA seq is better if you have a lot of samples to analyze.

[Benn]

Make a mouse model, preferentially with conditional knock-out of the gene/mutation, and see if the disease phenotype is present in these mice.