Dig labeled lna modified probes

Northern blots were performed using a DIG luminescent detection kit (Roche) according to the manufacturer’s instructions. Briefly, total RNA (2 μg) was mixed with RNA loading buffer (no. 9168, Takara) and heated at 65°C for 10 min. After cooling on ice for 10 min, the samples were loaded on an agarose (1.5%)-formaldehyde (2.2 M) gel at 60 V for 4 h. The separated RNAs were then transferred to a positively-charged nylon membrane (Thermo Fisher Scientific) and crosslinked with UV light. DIG-labeled LNA-modified probes (Exiqon) complementary to the target genes were hybridized to the membrane at 55°C overnight. The sequences of the probes were as follows: lnc-TSI, 5′-ACATCTCTTAATCAGCGAATCA-3′; ACTB, 5′-CTCATTGTAGAAGGTGTGGTGCCA-3′.

Ascidian embryos, larvae, and juveniles at 21, 34, and 42 hpf were collected and fixed in 4% paraformaldehyde (PFA) overnight at 4 °C. DIG-labeled LNA-modified probes (Additional file 5: Table S3) were designed and synthesized by Exiqon (Denmark). Whole-mount in situ hybridization was performed as previously described [37 (link)] at a temperature 20 °C below the probe melting temperature. Briefly, after fixation, samples were placed in PBST and washed four times at room temperature. Then, samples were treated with proteinase K at 37 °C for 20 min. After treatment, samples were re-fixed in 4% PFA and washed four times in PBST. After washing, samples were pre-hybridized in a prehybridization buffer for 2 h in a humid chamber. Subsequently, hybridization was conducted at hybridization temperature for 18 h using miRNA LNA probes in a hybridization solution. After hybridization, samples were washed in gradient saline-sodium citrate at the hybridization temperature. Signals of hybridization were detected using alkaline phosphatase-conjugated digoxigenin antibody (Roche) at a 1:2000 dilution. Samples were stained with BCIP/NBT and visualized under a microscope.