Perfecta sybr green fastmix green fluorescent dye

Primers were designed using MC2R, StAR and P450scc sequences previously published for rainbow trout (Table 1) [26 ] and were purchased from Integrated DNA Technolgies (Iowa, USA). The mRNA abundance was analyzed using the PerfeCTa SYBR Green FastMix green fluorescent dye (Quanta Biosciences, Gaithersburg, MD, USA) with the Bio-Rad C1000 Touch Thermal Cycler with CFX96™Real-Time System. The complete thermocyle was as follows: one cycle of 94°C for 2 min, 40 cycles [95°C for 30 sec, Annealing Temperature for 30 sec], 72°C for 10 min, followed by the melt curve. A melt curve (from 60–90°C in 0.5°C increments every 30 sec), along with the negative RNA and a negative cDNA control were used to ensure that a single product was amplified and that there was no contamination of the solutions. Copy number of each gene was determined by using relative standard curves, where the standards were made by serially diluting a pool (mix of 1 μl cDNA from each sample) of cDNA made from all the samples. All samples were assessed for the gene of interest, in duplicate, while the standard curve was run in triplicate. The relative transcript levels were determined by quantitative real-time PCR, as previously described [21 (link)] and each was normalized to the geometric mean of two reference genes, elongation factor 1 alpha and 18s ribosomal RNA (Table 1).

Field formulation TFM (Clariant SFC GMBH WERK, Griesheim, Germany) was used for all experiments [35% active ingredient dissolved in isopropanol; provided courtesy of Fisheries and Oceans Canada (DFO), Sea Lamprey Control Centre, Sault Ste. Marie, ON, Canada], and TFM exposure concentrations were verified using precision TFM standards provided by DFO. The Glucose Liquicolor® assay kit (Stanbio Laboratory, Boerne, TX, USA) was used to measure tissue and plasma glucose levels. High-binding 96 well plates (Immulon HB, VWR Canlab, Mississauga) were used for an in-house cortisol enzyme-linked immunosorbent assay (ELISA), used in conjunction with a cortisol monoclonal antibody conjugated to horseradish peroxidase (East Coast Bio, ME, USA). The RiboZol^TM used for RNA extraction was obtained from AMRESCO (Solon, OH, USA), while the DNaseI was purchased from Sigma-Aldrich and the first strand cDNA synthesis kit was from Invitrogen (Burlington, ON, CA). The PerfeCTa® SYBR® Green FastMix® green fluorescent dye used for real-time quantitative PCR (RT qPCR) was purchased from Quanta Biosciences (Gaithersburg, MD, USA), while the 96-well plates were obtained from Bio-Rad (Mississauga, ON, CA). All other chemicals were purchased from Sigma-Aldrich (MO, USA).