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Conservation, genomic synteny and expression timing of microRNA-34 family members in zebrafish. (A) Multiple sequence alignment of human (hsa) and zebrafish (dre) microRNA-34 (a, b, c) sequences. Identical nucleotides are marked with “*” in the consensus and in green. Nearly identical nucleotides (in 5 out of 6 microRNAs) are highlighted in yellow. The seed sequences are surrounded by red boxes. A cladogram showing phylogenetic relatedness of these microRNAs is shown next to microRNA names. (B) Synteny of genomic miRNA-34a regions in zebrafish and human. Start positions of the genes are marked with small rectangles (red for miR-34a and green for all other genes). (C) Synteny of miR-34b/c cluster location next to the btg4/BTG4 gene in zebrafish and humans. The miR-34b and miR-34c primary transcripts in both species are indicated with red rectangles and btg4/BTG4 exons are shown in green. MIR34B and MIR34C transcripts in human consist of a single exon shown by a rectangle with a black outline. Promoter regions are indicated with directed arrows. The opposite orientation of the promoters for the miR-34b/c cluster and btg4/BTG4 gene is also conserved from zebrafish to humans. (D) Quantitative real-time PCR analysis of the relative expression of miR-34a,b,c at different stages of zebrafish development. Triplicate biological samples of each stage and duplicate technical replicates were used for the analysis. The expression levels were normalized using 18S rRNA expression and the relative levels of all samples and genes were calculated relative to the level of miR-34a at 24 hpf. (E) Agarose gel analysis of semi-quantitative RT-PCRs of miR-34a,b,c transcripts at 48, 72 and 96 hpf stage zebrafish cDNAs synthesized with either random 9-mer or oligo-dT oligos.
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