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We assessed the expression stability of several messenger (m)RNAs and micro (mi)RNAs from liver and head kidney of rainbow trout Oncorhynchus mykiss using high‐throughput RNA sequencing (RNA‐seq) and miRNA‐seq data. Additionally, four commonly used reference genes and one small non‐coding RNA (u6) were also selected to identify ideal reference mRNAs and miRNAs for quantitative real‐time (qrt)‐PCR analysis of heat stress responses. GeNorm, NormFinder, BestKeeper and comparative ΔCt were employed for analysis of qrt‐PCR data to systematically assess the expression stability of candidate mRNAs and miRNAs and stability was ranked using geometric means. β‐actin and ef1‐α were the most stably expressed reference mRNAs in liver and head kidney, respectively and ssa‐mir‐26a‐5p and ssa‐mir‐462b‐5p were the most stably expressed miRNAs in these tissues. This is the first identification of appropriate reference mRNAs and miRNAs for qrt‐PCR analysis of O. mykiss under heat stress. [ABSTRACT FROM AUTHOR]

Rainbow trout (Oncorhynchus mykiss) is an economically significant freshwater-farmed fish worldwide, and the frequent outbreaks of infectious hematopoietic necrosis (IHN) in recent years have gravely compromised the healthy growth of the rainbow trout aquaculture industry. Fish skin is an essential immune barrier against the invasion of external pathogens, but it is poorly known about the role of circRNAs in rainbow trout skin. Therefore, we examined the expression profiles of circRNAs in rainbow trout skin following IHNV infection using RNA-seq. A total of 6607 circRNAs were identified, of which 34 circRNAs were differentially expressed (DE) and these DE circRNA source genes were related to immune-related pathways such as Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, Cytokine-cytokine receptor interaction, ubiquitin mediated proteolysis, and ferroptosis. We used qRT-PCR, Sanger sequencing, and subcellular localization to validate the chosen DE circRNAs, confirming their localization and expression patterns in rainbow trout skin. Further, 12 DE circRNAs were selected to construct the circRNA-miRNA-mRNA regulatory network, finding one miRNA could connect one or more circRNAs and mRNAs, and some miRNAs were reported to be associated with antiviral immunity. The functional prediction findings revealed that novel_circ_002779 and novel_circ_004118 may act as sponges for miR-205-z and miR-155-y to regulate the expression of target genes TLR8 and PIK3R1, respectively, and participated in the antiviral immune responses in rainbow trout. These results shed light on the immunological mechanism of circRNAs in rainbow trout skin and offer fundamental information for further research on the innate immune system and breeding rainbow trout resistant to disease., Competing Interests: Declaration of competing interest The authors declare that the study was no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)