Computational protocol: Sex steroid production associated with puberty is absent in germ cell-free salmon

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Protocol publication

[…] RNA was extracted from gonad and pituitary tissue using the iPrep™ Trizol® Plus RNA Kit (Thermo Fischer Scientific), according to the manufacturer’s instructions. Purified RNA was then treated with the turbo DNA-free kit (Ambion) to remove trace amounts of contaminating DNA. 500 ng of treated RNA served as input for reverse transcription reaction using the VILO cDNA synthesis kit (Invitrogen) for each sample. Previously published primers and hydrolysis probes specific for Atlantic salmon fshb, lhb , gnrhr4 , fshr, lhcgr , igf3, amh , cyp19a1a and bmp15l were used in this study. Furthermore, primers and probe sequences for cyp17a1, star and cyp11β were designed online (http://probes.pw.usda.gov/batchprimer3/) and are listed in Supplementary Table . The primer sequences for insl3 (Supplementary Table ) were designed using Primer Express 3.0 (Applied Biosystems); cloning and sequencing of salmon insl3 is described in Supplementary Note S.All qPCR assays were performed in duplicates, using 384-well optical plates on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) (male pituitaries) or QuantStudio 5 Real-Time PCR System (ThermoFisher Scientific) (testis, ovaries and female pituitaries) using default settings. One µl of a 1/20 (gonads) or 1/40 dilution (pituitaries) of cDNA was used in a 10 µl Fast Taqman qPCR reaction (ThermoFisher Scientific). In the case of star, cyp17a1, cyp11β and insl3, 1 µl of a 1/160 dilution of cDNA was used in a 10 µl Fast SYBR green qPCR reaction (ThermoFisher Scientific). For each qPCR assay, melting-curve analysis showed that only one product was generated. For each PCR plate, no-template controls were run for each gene. The relative gene expression levels for all genes were calculated using the comparative Ct (or 2−ΔΔCt) method. For each gene, all values were normalized to ef1a and calibrated to the average ΔCt of the immature WT group. […]

Pipeline specifications

Software tools BatchPrimer3, Primer Express
Application qPCR
Chemicals Steroids, Testosterone, Bone Morphogenetic Protein 15