Validation of Real-time PCR Reference Genes of Muscle Metabolism in Harvested Spiny-Cheek Crayfish (Faxonius limosus) Exposed to Seasonal Variation

SIMPLE SUMMARY: In the age of shrinking stocks and intensive animal production alternative raw materials are in the eye of the scientists. Here, we aimed to identify molecular tools to evaluate the quality of spiny-cheek crayfish meat. Results showed a set of genes that were steadily or deferentially expressed between seasons and sexes. Additionally, the authors revealed genes involved in molting cycle and muscle growth/atrophy of the crayfish. The suite of molecular tools developed in the study have potential for the profiling of crayfish raw material quality in the food sector. ABSTRACT: Real-time quantitative reverse transcription PCR (RT-qPCR) is a sensitive and broadly used technique of assessing gene activity. To obtain a reliable result, stably expressed reference genes are essential for normalization of transcripts in various samples. To our knowledge, this is the first systematic analysis of reference genes for normalization of RT-qPCR data in spiny-cheek crayfish (Faxonius limosus). In this study, expression of five candidate reference genes (actb, β-actin; gapdh, glyceraldehyde-3-phosphate dehydrogenase; eif, eukaryotic translation initiation factor 5a; ef-1α, elongation factor-1α; and tub, α-tubulin) in muscle samples from male and female F. limosus in spring and autumn was analyzed. Additionally, the most stable reference genes were used for accurate normalization of five target genes, i.e., tnnc, troponin c; ak, arginine kinase; fr, ferritin; ccbp-23, crustacean calcium-binding protein 23; and actinsk8, skeletal muscle actin 8. Results obtained using the geNorm and NormFinder algorithms showed high consistency, and differences in the activity of the selected actb with eif genes were successfully identified. The spring and autumn activities of the target genes (except ak) in the muscle tissue of males and females differed significantly, showing that both sexes are immensely involved in an array of breeding behaviors in spring, and females intensively recover in the autumn season. Characterization of first reference genes in spiny-cheek crayfish will facilitate more accurate and reliable expression studies in this key species.