Spatiotemporal expression of miRNAs in tissues undergoing metamorphosis. (A) Hierarchical clustering analysis of miRNA expression in four tissues undergoing metamorphosis from larval to pupal stages. Only miRNAs presenting signals higher than 1,000 in at least six specimens were scored as positive and subjected to cluster analysis. In all, data from 83 miRNAs were finally scored as expressed. Clustering analysis was also performed by CLUSTER 3.0/TreeViewsoftware based on array spot median center. (B) miRNAs were obviously differentially expressed in these tissues in microarray analysis. The colors represent relative expression for each miRNA, specifically, green, low; black, mean; and red, high. (C) Northern blotting analysis of miRNAs in four tissues undergoing metamorphosis. 5 S rRNA and U6 served as loading controls. Both microarray and Northern blotting analyses with alternative probes for bmo-miR281-5p, bmo-miR-10b-5p, bmo-miR-1, bmo-let-7a and bmo-miR-275 yielded reproducible results. Abbreviations: 7d IL5, Fifth-instar day 7 larvae; 0hr Sp, 0 hour spinning larvae; 0hr PPu, 0 hour prepupae; 1d Pu, day 1 pupae; HD, head; BW, body wall; SG, silk gland; MG, midgut; FB, fat body; 'a' and 'b' represent the average signals of individual probes printed at three points on each block.