Fig. 2.

Identification of Hand2-associated microRNAs by multiplex Taqman PCR. (A) miR-1 and miR-133a association was identified by multiplex PCR analysis of primary cardiomyocytes infected with lentiviruses expressing the GFP-Hand2 3′UTR-MS2 reporter. Six microRNAs, shown in blue, were abundantly expressed in heart, but did not associate with the Hand2 3′UTR probe. Results represent four independent experiments. Data are normalized to a U6 internal control represented on the array. (Mean ± SE are shown.) (B) Individual Taqman PCR confirmations of microRNA binding to probes containing the Hand2 3′UTR or reverse 3′UTR in primary cardiomyocytes. Hand2 3′UTR in correct and reverse orientation are shown in black and gray, respectively. Note scale differences in the two graphs. Results are representative of four independent experiments. (C) miR-133a interaction does not occur postlysis. Extracts from HEK293 cells expressing the MS2-tagged Hand2 3′UTR were mixed 1∶1 with extracts from differentiated C₂C₁₂ cells. No miR-133a binding was detected in the mixed extracts (left lanes). Differentiated C₂C₁₂ cells transfected with the MS2-tagged Hand2 3′UTR (middle lanes) or HEK293 cells expressing the MS2-tagged Hand2 3′UTR and transfected with miR-133a (right lanes) showed binding of miR133a in a 3′UTR-dependent manner. Blue and gray bars represent transcripts containing or lacking the Hand2 3′UTR, respectively. Similar results were obtained in three separate experiments.