Qiacube system 230 5

Prior to extraction, gill and kidney tissues were independently homogenized using a Qiagen TissueLyser LT (Qiagen, Valencia, CA, USA). A QIAcube system 230 V (Qiagen) was used to extract genomic DNA (gDNA), and total RNA from gill and kidney tissues using the DNeasy Blood and Tissue Kit (Qiagen) and RNeasy Plus Mini Kit (Qiagen), respectively, according to the manufacturer’s recommendations. Extracted gDNA and RNA were assessed for quality (A260:280 and A260:230 ratios) and concentration (ng μl⁻¹) using a NanoDrop ND1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). Integrity of RNA was assessed by electrophoresis on a 1% w/v agarose gel. A QuantiTect Reverse Transcription Kit (Qiagen) was used to synthesize cDNA from 1 μg of total RNA following the manufacturer’s protocol, with the exception that reaction volumes were scaled to a final volume of 40 μl. Fish intestine samples were digested using a modified ‘boiled-crude’ method of Palenzuela et al. (1999) (link): incubation at 56°C for 1–2 h with 180-μl buffer ATL (Qiagen) and 20-μl proteinase K to digest tissue, followed by heat denaturation at 85°C for 15 min, then dilution 1:100 prior to amplification in polymerase chain reaction (PCR).

Gill tissues were homogenized using a Qiagen TissueLyser LT (Qiagen) and RNA was extracted using the RNeasy Plus Mini kit with gDNA eliminator columns (Qiagen). All extractions were performed with a QIAcube instrument (Qiagen, QIAcube System 230 V) according to the manufacturer’s protocol. RNA concentration was assessed by measuring the A260:280 and A260:230 ratios on a NanoDrop (ND1000 Spectrophotometer, NanoDrop Technologies, Inc., Wilmington, DE, USA) and quality checked by electrophoresis. RNA (500 ng) was reverse transcribed to cDNA using QuantiNova Reverse Transcriptase kit (Qiagen) with integrated genomic DNA removal as per the manufacturer’s protocol. All cDNA samples were diluted 1:4 with RNAse free water.