RNAs at the different developmental WP1130 stages was confirmed. Direct cloning allowed us to identify several mature miRNAs sequences, such as bmo-miR-263a, bmo-miR-282, and bmomiR-317, which vary in their 59 and/or 39 ends on the same stem of their precursors. It is difficult to recognize accurate Core mfea 232.1 28.1 229.7 210 Hairpin mfeb Ch_ratioc 246.3 212 240 215 0.390.99 0.40.99 sequences of mature miRNAs solely by computational prediction and Northern blotting. Direct sequencing provides solid evidence, to the level of single nucleotide. For instance nine miRNAs in our dataset have nucleotide differences in 59 or/and 39 end of their sequences when compared the mature forms to their predicted sequences or homologs in closely related species. Moreover, we found that bmo-miR-2008 from the 59 stem of putative pre-miRNA S147 yields three different segments as mature miRNAs. Based on the numbers of sequenced clones per library, assuming unbiased cloning process, miRNA expression profiles in B. mori appeared stage-biased. We detected expressions of miRNAs in multiple developmental stages, and the frequency of any single miRNA appearing in sequenced libraries varied from one to seven. For instance, aside from a few commonly expressed miRNAs, such as bmo-miR-8, bmo-miR-13, bmo-miR-263a, bmo-miR-275, bmo-miR-279, bmo-282, bmo-miR-285 and bmo-miR-306, most of them were found development-related although some may be due to inadequate sampling that often results less reliable statistics. Furthermore, we noticed that the highest number of miRNAs, both in sorts and 
quantities, were detected in the library made from RNA isolated at molting larva stage –33 out of 77 sequences, representing 12 different miRNAs. Analyzing 15 mature miRNAs expression patterns and potential targets We analyzed the expression of 15 mature miRNAs from 14 different stages with stem-loop RT-PCR. The expression of bmo-miR-92 varied across the 14 developmental stages, highly expressed at five stages–pre-diapaused egg , diapause-broken egg , pupa and PPS with slightly 15976016 less significance –but lower in other stages, and the lowest at 25137254 diapaused egg stage. Bmo-miR-10 and bmo-miR-14 showed a synchronized trend where a sharp increase at trachea appearing stage , NLS, and MLS. BmomiR-31, bmo-miR-71, and bmo-miR-77 displayed a very similar expression pattern, higher expression at NLS; bmo-miR-31 and bmo-miR-77 are also expressed at a higher level at MLS than at other stages. Bmo-miR-8, bmo-miR-9a, and bmomiR-263a, similar to bmo-miR-1, showed a slight elevation after diapause-broken stage and kept a relatively constant level thereafter. In the case of bmo-miR-278 and bmo-miR-iab-4-3p, our results showed that they displayed distinct increases at MLS, late fifth-instar larva , PPS, and DS, SS, PS, respectively. The expression of bmo-miR-7 and bmo-miR-275 maintained at a relatively stable level in 14 development stages except bmo-miR-275 alone displayed a weak increase at both PPS and PS. Bmo-miR-13 showed fluctuating trend with its lowest level at LFLS. In general, most of the miRNAs exhibited the highest expression at NLS and MLS but the lowest expression at DS; these results are in accord with the cloning results for the MLS, where abundant miRNA expression was observed. We used the PITA program to identify the target genes of the 15 mature miRNAs with a default criterion and DDG#25 kcal/mol. From the long list of the target genes, we selected potential targets with high probability a