Validation of microarray-predicted changes in splice junctions using quantitative RT-PCR. Examples of alternative splicing patterns regulated by (a) insulin and (b) wingless signaling are shown. For each gene, a primer pair was designed to amplify a constitutive part of the transcript, thus monitoring general changes in transcription (exp). In addition, primer pair(s) in which one of the primers covers a splice junction were used to amplify and monitor changes in expression of particular isoforms, as indicated. Changes in splice junctions were evaluated relative to the change in gene transcription. RT-PCR results are presented as log2 ratio of eCp values obtained under conditions of pathway activation and controls. The corresponding Z-score values from the microarray prediction are indicated below the graphs for each event. Various classes of alternative splicing events are detected, including alternative first exons, alternative 5' or 3' splice sites, cassette and mutually exclusive exons and more complex patterns. In some cases, expression changes are not significant and alternative splicing changes are detected in the absence of significant changes in expression (for example, wdb, cg2201, trx, stat92E). In others, changes in splice junctions are clearly distinct from changes in expression (for example, cg14207) or even occur in the opposite direction (for example, babo). In some instances, changes in one splice junction probe monitoring a particular spliced isoform are not reciprocated by converse changes in probes monitoring the alternatively spliced product. This suggests the existence of additional processing pathways. Indeed, semi-quantitative RT-PCR using primers external to some of the alternatively spliced regions frequently detects the existence of additional, non-annotated isoforms (data not shown).