tissue: Whole brain Sex: Male maturity: Immature-1 genotype: LL
Treatment protocol
Not applicable
Growth protocol
Described in Debes, P. V., N. Piavchenko, J. Erkinaro, and C. R. Primmer, 2020 Genetic growth potential, rather than phenotypic size, predicts migration phenotype in Atlantic salmon. Proc. R. Soc. B Biol. Sci. 287: 20200867.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from wholee brain using a NucleoSpin RNA kit (Macherey-Nagel GmbH & Co. KG). The collected samples were transferred to tubes containing 1.4 mm ceramic beads from Omni International, along with Buffer RA1 and DDT (350 µl of RA1 and 3.5 µl of 1M DDT). Homogenization was performed using the Bead Ruptor Elite (Omni International) at a frequency of 30 Hz for a total of 2 min (in 6 cycles of 20 s each). The subsequent RNA extraction procedures adhered to the guidelines provided by the manufacturer, with the kit also including an integrated DNase stage to eliminate any remaining genomic DNA (gDNA). At the conclusion of this process, the RNA extracted from each individual sample was eluted using 50 µl of nuclease-free water. To assess RNA quantity, measurements were taken using the NanoDrop ND-1000 instrument (Thermo Scientific, Wilmington, DE, USA), while the quality of the RNA was evaluated through the employment of the 2100 BioAnalyzer system (Agilent Technologies, Santa Clara, CA, USA). The RNA integrity number (RIN) exceeded 7 for all samples.
Label
No label
Label protocol
Not applicable
Hybridization protocol
One hundred nanograms of total RNA were hybridized with the NanoString Technologies nCounter a Custom made Panel containing 342 unique pairs of 100bp reporter probes and biotin-labeled capture probes, including internal reference controls. Overnight hybridization occurred for 20 hours at 65°C, then reactions were held at 4°C until they were placed on the nCounter Prep Station. Wash protocol: Removal of excess probes with magnetic bead purification was performed on the nCounter Prep Station (software v4.0.11.2) on the High Sensitivity assay. Briefly, the probe-mRNA structure was affinity purified by its 3’ end to remove excess reporter probes, then by its 5’ end to remove excess capture probes. Once unbound probes were washed away, the tripartite structure was bound to the streptavidin-coated cartridge by the biotin capture probe, aligned by an electric current (negative to positive), and immobilized. Photobleaching and flurophore degradation was prevented with the addition of SlowFade.
Scan protocol
After hybridization, the sample strip was transferred to the nCounter Prep Station for purification and immobilization in a sample cartridge. After immobilization, the cartridge was analyzed with the nCounter Digital Analyzer by taking images of the immobilized fluorescent reporters. The number of images taken corresponded to the number of reporters counted. The raw count data was exported as a csv file.
Description
Nanostring data analysis on mRNA isolated from the brain of Atlantic salmon (S-salar) in different maturation status with different vgll3 genotypes (EE or LL)
Data processing
RCC files containing raw counts for 342 genes provided from nanostring were loaded into nSolver Analysis Software 4.0 and normalized for housekeeping genes and positive controls.
