Deletion of Dicer1 reduces mature miRNA level in platelets. (A) Schematic representing Pf4-cre recombinase-mediated deletion of Dicer1. LoxP sites flanking the second RNase IIIb domain of Dicer1 mediate its removal in the presence of Cre recombinase, which is under control of the MK and platelet-specific Pf4 promoter. (B) Real-time PCR amplification of platelet RNA from Dicer1^Pf4Δ/Pf4Δ or Dicer^fl/fl mice. The primers are designed to amplify within the Dicer RNase IIIb domain (top) or flank the Dicer RNase IIIb domain (bottom). Note that, in the Dicer1^Pf4Δ/Pf4Δ mice, the transcript is present in platelets, but truncated. (C) Western blots for Dicer1 and Gapdh in platelets from Dicer^fl/fl (lanes 1 and 3) or Dicer1^Pf4Δ/Pf4Δ (lanes 2 and 4) mice. (D) Dicer activity assay to monitor the processing of ³²P-labeled human pre–let-7a into mature let-7a using platelet lysates (or no lysate [–]; lane 1) from Dicer1^Pf4Δ/Pf4Δ (lane 3) or Dicer1^fl/fl littermate controls (lane 2). (E) Heat map depicting the clustering and relative fold changes of mature miRNAs, changed twofold or more, in platelets from Dicer1^Pf4Δ/Pf4Δ compared with Dicer1^fl/fl littermates (n = 3 mice per group. Each lane represents data from a single mouse/array for a total of 6 microarrays).