Identification of novel mosquito miRNAs. Primer extension analysis was used to confirm the expression of A) three novel Ae. albopictus miRNAs and C) one novel Cx. quinquefasciatus miRNAs. Total RNA was isolated from C7/10 cells or Cx. quinquefasciatus mosquitoes as described in Figure 3 and Methods. B) and D) Predicted pre-miRNA stem-loop structures for each novel miRNA. Ae. albopictus miRNAs were mapped to the Ae. aegypti genome, and therefore may not reflect the actual pre-miRNA structures. Mature miRNA sequences are shown in red, while corresponding miRNA* sequences identified in each library are shown in blue. For miR-2951, asterisks indicate additional 5' nucleotides present in a lower percentage of the reads mapping to each miRNA compared to the canonical sequence annotated in Tables 1 and 2. Ae. albopictus miR-2951 differs by one nucleotide from the Ae. aegypti genome. E) Pre-miRNA structures for two novel orthologous miRNAs mapping to the A. gambiae genome. The predicted mature miRNAs, based on sequence conservation, are shown in red.