Figure 3

Verification of 3′-oligouridine Addition in Cleaved HMGA2 mRNA Fragments Targeted by miR-142-3p.

(a) 3′-oligouridine additions on 5′-fragments of cleaved HMGA2 mRNA by miR-142-3p activity in HeLa cells by U-track–specific SLA–RT-PCR were detected by agarose gel electrophoresis. An SL-RT primer with 5′-AAAAAA-3′ (6U-SL-RT) as probe was used as the RT primer to randomly prime cleaved mRNA fragments in oligouridylated forms. The universal PCR primer and a 5′-PCR primer specific to HMGA2 mRNA were used to amplify possible 3′-oligouridine–modified fragments and several 6U-SLA–RT-PCR amplicons, termed oligo-thymine-specific PCR fragments (TSPFs), were detected on the agarose gel. MK: 0.03 μg of 1 kb DNA ladder. (b) 3′-oligouridine addition was verified by DNA sequencing. The 100-bp TSPF was dissected from the agarose gel and analysed by automated DNA sequencing.