Submitted to: Comparative Biochemistry and Physiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/14/2008
Publication Date: 7/22/2008
Citation: Small, B.C., Murdock, C.A., Bourgeois, A.L., Peterson, B.C., Waldbieser, G.C. 2008. Stability of Reference Genes for Real-Time PCR Analyses in Channel Catfish (Ictalurus punctatus) Tissues Under Varying Physiological Conditions. Comparative Biochemistry and Physiology 151:296-304. Interpretive Summary: Improved production characteristics, such as faster growth, improved reproductive efficiency, and reduced stress tolerance, are needed to keep U.S. farm-raised catfish production competitive in today’s global economy. Technological advances have made the measurement of candidate genes, those thought to regulate important production characteristics, relatively easy using real-time PCR. Results of real-time PCR assays, however, must be normalized to make appropriate comparisons between genetically distinct individuals and groups of fish. Here, we report the evaluation and validation of several potential normalizers to address what regulates catfish growth, reproduction, and stress tolerance. The results of this research provide a basis for interpreting gene expression in channel catfish and enhance our ability to identify the genes responsible for traits of economic importance to U.S. farm-raised catfish producers.
Technical Abstract: Real-time PCR is a highly sensitive, relatively easy to perform assay for quantifying mRNA abundance. However, there are several complexities built into the assay that can affect data interpretation. Most notably, the selection of an appropriate internal control for normalization is essential for expression data interpretation. In this study we investigated the suitability of seven commonly used genes [18S ribosomal RNA (18S), alpha tubulin (TUBA), beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 alpha (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), and RNA polymerase II polypeptide B (POLR2B)] as potential quantitative references for normalizing real-time PCR data generated in the study of channel catfish physiology. Gene expression and stability were evaluated among 15 channel catfish tissues and within physiologically-relevant tissues in response to experimental manipulation (i.e. LHRH injection, fasting, and acute stress). Expression of the seven candidate reference genes varied across all tissue types tested, indicating that none of the genes could suitably serve as reference genes for cross tissue comparisons. Experimentally altering the physiological state of the fish differentially affected expression of the various reference genes depending on experimental design and tissue type, with 18S unaffected by the experimental treatment in all tissues examined. For example, the selection of a differentially expressed gene, GAPDH, as opposed to 18S, to normalize hepatic growth hormone receptor during fasting resulted in misinterpretation of the data. These results reveal the importance of providing comprehensive details of reference gene validation when publishing real-time PCR results, with this manuscript serving as a basic guideline for reference gene selection in channel catfish research.