Detection of hav-miR-N1-3p from plasmid-expressed precursor miRNA (pre-miRNA). (A) Construction of miRNA expression plasmid. Each pre-miRNA sequence was cloned into a pcDNA6.2-GW/EmGFP-miR expression vector. The vector with scrambled sequences was used as a negative control. (B) KMB17 and HEK293T cells were transfected with miRNA expression plasmid, and strong green fluorescence was observed in both transfected cell lines (100 ×). (C) Detection of mature miRNA hav-miR-N1-3p expression. Small RNA was harvested at 24 hours post-transfection, and mature hav-miR-N1-3p was detected by stem-loop RT-PCR in both transfected cells. (D) Plasmid-expressed hav-miR-N1-3p was reduced in Dicer-deficient KMB17 and HEK293T cells compared with Dicer-non-deficient negative control cells. Significant difference in expression levels were detected by Student’s t-test (*P < 0.05 and **P < 0.01).