Reverse Genetics Screen in Zebrafish Identifies a Role of miR-142a-3p in Vascular Development and Integrity
A –Relative quantification of miR-142a-3p in GFP positive (+) and GFP negative (–) cells of 2 dpf Tg(fli1:EGFP, gata1a: dsRed) zebrafish embryo by real-time PCR. Total RNA was isolated from 2 dpf FAC sorted GFP (–) and GFP (+) cells of 2 dpf Tg(fli1:EGFP, gata1a: dsRed) zebrafish embryos. 200 ng of total RNA was used for preparing cDNA using QuantiMir kit, SBI, USA and diluted by 1∶1 for RT-PCR assay. The assay has been performed using ΔΔCT method (ΔΔCT = (CT miRNA - CT reference RNA) - (CT calibrator - CT reference RNA) as described previously . U6 and beta-actin was used as calibrator and reference control respectively. Data collected from 3 independent experiments is represented as mean fold change ± SD. B – Schematic alignment of miR-142a-3p MO with pre-miR-142a. Mature miR-142a-3p sequence is in red color. C - Relative quantification of mature miR-142a-3p in non-injected control (NIC) and embryos injected with 200 µM miR-142a-3p MOs at 2 dpf. The assay was performed as described by manufacturer (QuantiMir kit, SBI, USA). The relative expression of miR-142a-3p was normalized to miR-26a. Data collected from 3 independent experiments is represented as mean fold change ± SD. D - G - Representative image of 28–30 hpf Tg(fli1:EGFP, gata1a: dsRed) zebrafish embryos. Inset displaying intersegmental vessels from non-injected control and 200 µM miR-142a-3p morpholino injected embryos. Images are arranged in a lateral view and inset displaying 11–15 intersegmental vessels from the trunk region. The images are taken at 5× and 10× magnifications. Arrowheads indicate regions with vascular defects. H - Bar graph showing embryos with inter-segmental vessel defects in non-injected control (NIC) and 200 µM miR-142a-3p morpholino injected embryos at 28–30 hpf.