Rnable reagent

The expression profiles of urp1 and urp2 genes in adult zebrafish were examined by RT-PCR using gene-specific primers of each transcript (see S1 Table for the primer sequences). Total RNA was extracted from various tissues, including brain, spinal cord, eyes, skin, muscle, heart, spleen, gill, gas bladder, intestine, liver, kidney, ovary, and testis, following the protocol provided with RNAble reagent (Eurobio, Les Ulis, France) and further purified by using RNAeasy Plus Mini kit (Qiagen, Courtabœuf, France). For each tissue, 242 ng of total RNA was reverse transcribed by using ImProm-II Reverse Transcription System (Promega, Charbonnières, France). PCR was carried out using a MyCycler thermal cycler (Bio-Rad, Marne la Coquette, France) for 25 cycles (denaturation: 95°C, 2 min; annealing: 56°C, 30 sec; extension: 72°C, 30 sec). The zebrafish β-actin gene was amplified as an internal control to verify the integrity of all cDNA samples (primer sequences are given in S1 Table). Negative controls were performed without cDNA template. All PCR products were electrophoresed in 1% agarose gel stained with SybrSafe (Invitrogen, Pontoise, France) then detected under UV light with the Gel Doc XR⁺ System (Bio-Rad).

The rim of third ventricle of adult mice was dissected under a binocular microscope, snap-frozen in liquid nitrogen and stored at −80°C until processed. Total RNA was extracted based on the protocol provided with the RNAble reagent (Eurobio, Les Ulis, France). Concentration of total RNA was measured, and RNAs were stored in Tris 10 mM/EDTA 0.1 mM (pH 7.4) at −80°C. To quantify mRNAs 1 µg of total RNA was reverse-transcribed using High capacity cDNA Reverse Transcription kit (Applied Biosystems, Courtaboeuf, France). Control reactions without reverse transcriptase were done in parallel. Primers for the detection of NgR (Taqman gene expression assays, references: Mm00710554_m1) and control assay GAPDH (Mm99999915_g1) were purchased from Applied Biosystems. Direct detection of the PCR product was monitored by measuring the increase in fluorescence generated by the TaqMan probe (NgR, GAPDH) as described previously by Decherf et al. (2010) [38] (link).