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Validation After you have identified genes with altered expression in your experimental group, the expression levels of a sub-set of these should be validated. Most journals will not publish without some validation of the gene sets identified in microarray studies. Real-time PCR (two systems available on site) Northerns Quantitative RT-PCR (multiplex with internal standard best option for RT-PCR) NB: Probe match on NetAffx (Affymetrix website) can be used to check your oligonucleotide against the target sequence. NetAffx also contains all sequence information for oligonucleotides used on the GeneChip™ arrays. These can be used in your validation studies as the oligonucleotides have been selected for optimal, specific hybridization to the target sequence. The signals for each oligonucleotide and its paired mismatch are in the CHP files associated with each sample. This allows you to check the efficiency and specificity of hybridization for each oligonucleotide to each of the samples that you have analysed. You may wish to extend your studies to examine protein expression levels of the genes identified as ‘interesting’ in your microarray study (e.g. Westerns, IHC, etc).
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