Agencourt rnadvance tissue total rna purification kit

Total RNA from the gills (N=6 fish per treatment group; three per replicate tank) was isolated from the RNAlater^®-preserved samples using the Agencourt RNAdvance™ Tissue Total RNA Purification Kit (Beckman Coulter Inc., CA, USA). All samples had an RNA Integrity Number (RIN) above 8.4 as evaluated by the Agilent^® 2100 Bioanalyzer™ RNA 6000 Nano Kit (Agilent Technology Inc., Santa Clara, CA, USA). The microarray analysis was performed using a custom-designed 15K Atlantic salmon DNA oligonucleotide microarray SIQ-6 (Agilent Array, ICSASG_v2), and all re-agents that were used were from Agilent Technologies. The One-Color Quick Amp Labelling Kit (CA, USA) was used for RNA amplification and Cy3 labelling using 110 ng of RNA template per reaction. Gene Expression Hybridization Kits were used for the fragmentation of labelled RNA. This was followed by a 15-h hybridization in a 65°C oven with a constant rotational speed of 10 rpm. Thereafter, the arrays were successively washed with Gene Expression Wash Buffers 1 and 2 and scanned using the Agilent SureScan Microarray Scanner. Pre-processing was performed in Nofima’s bioinformatics package STARS (Salmon and Trout Annotated Reference Sequences) (34 (link)).

An Agencourt RNAdvance™ Tissue Total RNA Purification Kit (Beckman Coulter Inc., CA, United States) with the aid of Biomek 4000 automated workbench station (Beckman Coulter, Inc., CA, United States, Appendix IV) was used to isolate the total RNA from the samples. The isolated RNA was quantified using NanoDrop 8000 Spectrophotometer (ThermoFisher Scientific, United States). The quality of RNA samples was considered good if the nucleic acid-protein ratio was between 1.9–2.1. Consequently, the complementary DNA (cDNA) was synthesised from 500 ng template RNA using a High-Capacity cDNA Reverse Transcription Kit (Beckman Coulter, Inc., CA, United States) in a 20-μL reaction volume and thermocycling was carried out in a Veriti™ 96-Well Thermal Cycler 7 (Applied Biosystems, California, United States) with the following conditions: 10 min at 25°C, 30 min at 37°C, and 5 min at 95°C.

Liver, spleen and skeletal muscle were stored in RNAlater (Ambion) before RNA was isolated using an Agencourt^® RNAdvance™ Tissue Total RNA Purification Kit (Beckman Coulter Inc.) according to the manufacturer’s protocol. NanoDrop 1000 Spectrophotometer (ThermoFisher Scientific) was used to evaluate the RNA quality and quantity. The quality of the RNA was further assessed with an Agilent^® 2100 Bioanalyzer™ RNA 6000 Nano kit (Agilent Technology Inc.). All RNA samples had RNA Integrity Number value higher than 8.
cDNA was synthesised from 1000 ng total RNA in a 20 µl reaction volume using TaqMan Reverse Transcription Reagents (Applied Biosystems, ThermoFisher) with random hexamers as reaction primers. The thermocycling conditions included incubation at 25°C for 10 min followed by 37°C for 30 min and 95°C for 5 min.