Rna tissue mini kit

Total RNA was extracted and treated with BioRobot^® EZ1 and RNA Tissue Mini Kit (Qiagen, Hilden, Germany) as previously described by Le, Shao (48). RNA quantity and integrity were validated using a NanoDrop ND-1000 UV—vis Spectrophotometer (NanoDrop Technologies, Wilmington, USA) and Agilent 2100 Bioanalyzer and RNA 6000 Nano LabChip kit (Agilent Technologies, Palo Alto, USA) respectively. All samples had 260/230 and 260/280 ratios above 2.0 and 2.2 respectively. The average RNA integrity number (RIN) of all samples was 7.9±0.7. Sequencing and library preparation were performed by the Norwegian Sequencing Centre (www.sequencing.uio.no). DNA libraries were prepared as previously described by [46 (link)] using 90 ng total RNA input to the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, California, USA). For multiplexing, standard Illumina adaptors were used. The libraries were sequenced using the NextSeq Illumina platform (Illumina, San Diego, California, USA) according to the manufacturer’s instructions, generating single end 75bp read libraries with an average library size of 25±6 million reads. Raw reads were submitted to the gene expression omnibus https://www.ncbi.nlm.nih.gov/geo/ (accession number GSE129459).

Zebrafish embryos (pools of 40 per sample) were homogenized using ceramic beads CK28 and a Precellys 24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). Total RNA was extracted using RNA Tissue Mini Kit (Qiagen, Hilden, Germany) and the BioRobot EZ1, treated with DNase according to the manufacturer’s instructions and eluted in 50 μL RNase-free MilliQ H₂O. Quality and integrity of extracted RNA were validated with the NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, United States) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, United States).
PCR primer sequences used for quantification of the transcriptional levels of the evaluated genes are shown in Table 1. A two-step real-time RT-PCR protocol previously described by Olsvik et al. (2014) (link) was used to quantify the transcriptional levels of the selected genes. Mean normalized expression (MNE) of the target genes was determined using a normalization factor based upon actb, eef1a1, and uba52 (M < 0.59), as calculated by the geNorm software (Vandesompele et al., 2002 (link)).

Tissues from Atlantic salmon were homogenized with the Precellys 24 homogenizer by using ceramic beads CK28 (Bertin Technologies, Montigny-le-Bretonneux, France). Total RNA was extracted using the BioRobot EZ1 and RNA Tissue Mini Kit (Qiagen, Hilden, Germany) and treated with DNase according to the manufacturer’s instructions and eluted in 50 μL RNase-free MilliQ H₂O. The RNA was then stored at −80 °C before further processing. RNA quality and integrity were assessed with the NanoDrop ND-1000 UV–Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The 260/280 and 260/230 nm ratios in liver were 2.11 ± 0.01 and 2.22 ± 0.01, respectively (N = 38, mean ± SEM). The RNA 6000 Nano LabChip kit (Agilent Technologies, Palo Alto, CA, USA) was used to evaluate the RNA integrity of the samples. The RNA integrity number (RIN) was 9.3 ± 0.1 (N = 12) in liver (mean ± SEM).