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. 2020 Nov 23;11:569412. doi: 10.3389/fmicb.2020.569412

FIGURE 2.

FIGURE 2

Identification of GLV miRNA1. (A) The hairpin structure sequence containing GLV miRNA1. (B) The polymerase A–based RT-PCR of GLV miRNA1, 1: DNA ladder, 2: amplicons of GLV miRNA1. (C) Splinted ligation Northern blot of GLV miRNA1, M: oligo = 40 nt (5′-CGT ACG TAT ATA TTT ATG GCG GCG CGA TGC TAC TCT CTC G-3′ DIG) + 20 nt (5′-CGT ACG TAT ATA TTT ATG GC-3′ DIG), 1: probe (5′-phosCGC TTA TGA CAT TC-3′ DIG 14 nt), 2: bridge (5′-GAA TGT CAT AAG CGC GGC TGC TGT AAC AGG ACC AGA-3′ 36 nt), 3: probe and bridge, 4: probe + bridge + G. duodenalis RNA (virus-free), 5: probe + bridge + G. duodenalis RNA(with virus). (D) Existence of pre-GLV miRNA1 by primer extension M: 20 bp marker (TaKaRa), 1: products of primer extension. (E) GlDcr is required for processing the mature GLV miRNA1, 1: G. duodenalis trophozoites total RNA, 2: pre-GLV miRNA1 RNA digested with GlDcr, 3: pre-GLV miRNA1 RNA digested without GlDcr.