Submitted to: Comparative Biochemistry and Physiology
Publication Type: Abstract Only
Publication Acceptance Date: August 9, 2005
Publication Date: February 13, 2006
Citation: Small, B.C., Murdock, C.A., Bourgeois, A.L., Peterson, B.C., Waldbieser, G.C. 2006. 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. Technical Abstract: Quantitative real-time RT-PCR represents a sensitive and convenient method for measuring mRNA levels from a given sample. However, one potential pitfall of using this technique comes when choosing a suitable reference gene(s) for normalization. Normalization of expression to an internal standard is critical for any useful comparisons of expression levels one may wish to make between different tissue samples. Recent studies have suggested that the expression levels of many of the commonly used reference genes are not uniform, but instead can vary depending on treatment conditions, disease states, and tissue/cell types. The use of real-time RT-PCR has become increasingly popular in studies using aquaculture species, including channel catfish (Ictalurus punctatus). In the reported study a total of seven commonly used reference genes [18S, alpha-tubulin, beta-actin, beta2-microglobulin (beta2M), elongation factor1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and RNA polymerase II (RNAPII)] were investigated. Basal expression levels of all seven reference genes were determined for 15 different types of tissue (i.e., hypothalamus, pituitary, ovary, testis, head kidney, trunk kidney, pancreas, stomach, intestine, spleen, liver, gall bladder, heart, gill, and muscle). The expression data for each reference gene between these 15 tissues revealed 18S to be the gene with the most constant level of expression, followed by alpha-tubulin, beta-actin, beta2M, RNAPII, GAPDH, and EF (respectively). Further analyses of expression under different physiological conditions were conducted in tissues within the immune, somatotrophic, stress, and reproductive axis of channel catfish. For example, changes in expression of the selected reference genes were investigated in the hypothalamus-pituitary-head kidney axis in response to low-water stress. All reference genes, with the exception of RNAPII in the head kidney, were found to be suitable for normalization within the three tissues examined in response to stress. In conclusion, the results of these studies may serve as a guide for the selection of an appropriate reference gene for mRNA expression studies in channel catfish. It appears that no single reference gene is optimal across tissue types and physiological conditions. Therefore the choice of an internal standard (or multiple standards) for normalization should be determined based on the tissues of interest and experimental conditions.