Validation of Reference Genes for Real-Time Polymerase Chain Reaction Studies in Atlantic Salmon

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Abstract
Optimization of reference genes for real-time polymerase chain reaction (PCR) studies in fish is strongly needed. We systematically tested β-actin ( ACTB ), 18S rRNA ( 18S ), β_2-microglobulin ( B2M ), elongation factor 1-α ( EF1A ), RNA polymerase I and II ( RPL1/2 ), and glycerol 6-phosphate dehydrogenase ( G6PDH ) for stability in salmon immune relevant tissues and kidney cells (ASK) infected with infectious salmon anemia virus (ISAV), plus in tissues from fish fed thia fatty acids. Transcription of all genes was unchanged in infected and thia fatty acid-treated tissues versus normal tissues. Between tissues, 18S and EF1A were most stable, RPL1 and RPL2 were intermediate, and G6PDH and ACTB and B2M were the least stable. However, only 18S had constant expression in infected cells; the rest significantly down-regulated. Implications of this finding were demonstrated when normalizing major histocompatibility complex (MHC) class I expression in ASK. Software predictions supported a proper normalization is obtained combining 18S, EF1A , and RPL1 in vivo, but for in vitro viral infection assays we recommend using 18S .