Functional investigation of the tie-1AS lncRNA. (A) The RNA duplex was examined by ribonuclease protection assay. The zebrafish embryos at 24 hpf were digested with 0.25% trypsin into single cells, from which cytoplasmic and nuclear RNA was extracted. The RNAs were treated with DNase I and RNase A followed by RT-PCR. Primers used are as follows: lanes 1 and 4, P1 and P2, amplicon size 114 bp; lanes 2 and 5, P3 and P4, amplicon size 133 bp; and lanes 3 and 6, β-actin, amplicon size 147 bp. (B) Overexpression of tie-1AS lncRNA down-regulates the expression of tie-1. *P < .05. (C) No effect on the expression of tie-2. The zebrafish embryos were injected at the 1-cell stage with 150 pg of tie-1AS mRNA, and quantitative PCR was performed at 24 hpf. (D) Chemical treatment of zebrafish embryos with α-amanitin shows dose-dependent sensitivity to tie-1AS lncRNA and tie-1 (E) transcription.