Spatial expression patterns of miRNAs in fifth-instar day 3 larvae. (A) Hierarchical clustering of miRNAs based on the normalized microarray signal values in different tissues. The highest signal was 79,375.67 (miR-1 in female head), and the average signal was 3,228.516. Only miRNAs presenting signals higher than 1,000 in at least two specimens were saved as positive and subjected to cluster analysis. In all, 67 unique miRNAs were finally selected as 'confirmed.' CLUSTER 3.0/TreeView software was used for clustering analysis with the median center in array spots. (B), (C) and (D)Northern blotting analysis of miRNAs in multiple tissues of fifth-instar day 3 larvae. (B) Decade Markers (Ambion) were applied in our Northern blotting experiments to estimate miRNA sizes. (C) Initially, we only examined 12 tissues using Northern blotting, and investigated the whole silk gland. (D) For a better comparison with microarray results, we examined the 18 tissues (organs) corresponding to those examined by microarray. The signal bands displaying sex differences are boxed, and those showing some differences from microarray results are underlined. 5 S rRNA and U6 were applied as loading controls. Abbreviations: HD, head; BW, body wall; ASG, anterior silk gland; PSG, posterior silk gland; MG, midgut; FB, fat body; OV, ovary; TE, testis; HC, hemocyte; MT, malpighian tubule; f, female; m, male; TN, tissue number. lowercases 'f' and 'm' in parentheses of (A) are female and male, respectively; 'a' and 'b' represent the average signals of each probe printed at three points on individual blocks.