Nanodrop nd1000

RNA samples (N = 5–6) from 24H-cultured myogenic precursors from juvenile trout (JT), from 3 to 4 weeks fasted juvenile trout (FJT) and from adult trout (AT) were used for labelling and hybridization. For each sample, 150 ng of RNA was Cy3-labelled according to the manufacturer’s instructions (One-Color Microarray-Based Gene Expression Analysis (Low Input Quick Amp Labeling) Agilent protocol). Briefly, RNA was first reverse transcribed, using a polydT-T7 primer, Cy3 was incorporated by a T7 polymerase-mediated transcription and excess dye was washed using an RNeasy kit (Quiagen). The level of dye incorporation was evaluated using a spectrophotometer (Nanodrop ND1000, LabTech). 600 ng of labelled cRNA was then fragmented in the appropriate buffer (Agilent) for 30 min at 60 °C before dilution (v/v) in hybridization buffer. Hybridizations were performed in a microarray hybridization oven (Agilent) for 17 h at 65 °C, using two Agilent 8 × 60 K high-density oligonucleotide microarray slides. Following hybridization, the slides were rinsed in gene expression wash buffers 1 and 2 (Agilent).

RNA isolation was performed using the RNeasy Mini-kit according to the manufacturer’s instructions, and RNA concentration was determined using a NanoDrop ND-1000 (Labtech, East Sussex, UK). The quality of the RNA was determined using the Agilent RNA 6000 Nano Kit according to the manufacturer’s instructions, with samples analysed using the Agilent 2100 bioanalyzer (Agilent, Berkshire, UK).

Independent RNA isolations were carried out for each gastrocnemius muscle sample. Total RNAs were extracted using the RNeasy Fibrous Tissue Mini Kit (Qiagen) following the manufacturer’s conditions. RNA concentrations were measured using a NanoDrop ND-1000 (LabTech, Ringmer, UK), and RNA quality was verified by 1% agarose gel electrophoresis. One μg total RNA was used as a template for single-strand cDNA synthesis using High-Capacity cDNA RT Kit (Applied Biosystems, Foster City, CA, USA) in a total volume of 20 µL containing 1 X RT buffer, 4 mM dNTP mix, 1 X random primers, 50 U reverse transcriptase and 20 U RNase inhibitor. The primer sequence is reported in Table 1. The reverse transcription reactions were run as follows: 25 °C for 10 min, 37 °C for 120 min, and 85 °C for 5 s. PCR was carried out in a final volume of 20 µL containing 10 µL Power SYBR Green PCR Master Mix (Applied Biosystems), 0.4 µL of each primer at 10 pmol/µL, and 2 µL of the cDNA solution. qRT-PCR amplification was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad, Marnes-la-Coquette, France) with the following thermal cycler conditions: 15 min at 95 °C, followed by 45 cycles of 15 s at 95 °C, and 1 min at 60 °C. Raw data were analyzed using CFX Maestro (Bio-Rad) and compared by the ΔΔCt method. Results are expressed relative to the housekeeping gene (Actb) transcript quantity.

Hippocampal gene expression was measured, as previously described⁽⁸ (link)^,16 (link)⁾. Briefly, RNA was extracted using TRIzol reagent (Invitrogen). RNA concentrations were determined using a Nanodrop ND-1000 (Labtech). cDNA were synthesised from 1 µg of RNA with ImPromII reverse transcriptase (Promega), using oligodT and random primers (Promega).
Real-time PCR was performed using the LightCycler 480 system with a ninety-six-well format (Roche Diagnostics). The forward- and reverse-primer sequences and the amplicon size for glyceraldehyde-3-phosphate dehydrogenase (Gapdh), Ngf and Bdnf are summarised in Supplementary Table S2. Gapdh was used as the reference gene, since its expression level was unaffected under our experimental conditions.
Quantification data were analysed using LightCycler 480 Relative Quantification software (version 1.5). Therefore, the results are expressed as the target:reference ratio divided by the target:reference ratio of the calibrator. In our case, the calibrator was chosen among the adult mice.

Liver and white muscle samples after 36 weeks were homogenised in Trizol reagent (Invitrogen, Carlsbad, CA, USA) using the Precellys 24 (Bertin Technologies, Montigny-le-Bretonneux, France). The total RNA was then extracted according to the Trizol manufacturer’s instructions (n = 9). The concentration of extracted RNA was analysed using a spectrophotometer (Nanodrop ND1000, LabTech) by measuring absorbance at 260 nm and quality of RNAs was checked with Bioanalyzer (Agilent Technologies, Kista, Sweden).

Whole yolk-sac fry tissues were individually homogenized in ice using an Ultra Turrax homogeniser (T25 basic IKA-WERKE) at the speed setting of 4 for 10 s until they were homogeneous. Total RNA, DNA, and protein were extracted from whole yolk-sac fry (n = 16 per condition) using the QIAGEN AllPrep DNA/RNA/Prot Preparation Kit according to the manufacturer’s recommendations (Qiagen). The concentration of extracted RNA was analyzed using a spectrophotometer (Nanodrop ND1000, LabTech) by measuring absorbance at 260 nm. The quality of RNAs was checked with a Bioanalyzer (Agilent Technologies, Kista, Sweden), and 12 samples for each condition were selected for further analyses according to the RIN (RNA integrity number).

Various tissues were sampled into 2 mL EP tubes with 1 mL trizol (Invitrogen, Carlsbad, CA, USA), and RNA was extracted following the manufacturer’s protocol. The concentration of every samples’ RNA was determined using spectrophotometry Nanodrop ND1000 (LabTech, Beijing, China), and the integrity of RNA was examined using 1% agarose gel electrophoresis. For each sample, the equal mass of total RNA (1000 ng) was used for cDNA synthesis using the Hifair III 1st Strand cDNA Synthesis SuperMix for the qPCR set (YEASEN, Shanghai, China). Next, the cDNA was diluted three times for further qPCR analysis. qPCR was performed using the qTOWER3G PCR system (Analytik Jena, Jena, Germany) using Hieff qPCR SYBR GREEN Master Mix (YEASEN) under the following conditions: 95 °C for 5 min, then cycled 40 times at 95 °C for 10 s, at 58 °C for 30 s [50 (link)]. The qPCR primer sequences used in this experiment are provided in Table 1.