Both miRNA and miRNA* species can be detected in total RNA. RNA was analyzed from D. melanogaster cells at different stages: E, 0–24 h embryos; LP, 3rd instar larvae and mixed pupae; A, adult males and females; S, S2 cells. Each blot was sequentially hybridized, stripped and rehybridized to detect a miRNA* species, its partner miRNA species, then 2S rRNA (the exception is miR-10*, whose companion 2S blot is shown in Supplementary Fig. 1). Arrows indicate the mature 21–24 species and brackets indicate the pre-miRNA hairpin.