Stability of reference genes for real-time PCR analyses in channel catfish (Ictalurus punctatus) tissues under varying physiological conditions.
作者： Brian C. Small , Christopher A. Murdock , A. Lelania Bilodeau-Bourgeois , Brian C. Peterson , Geoffrey C. Waldbieser
DOI: 10.1016/J.CBPB.2008.07.010
关键词:
摘要: Real-time PCR is a highly sensitive, relatively easy to perform assay for quantifying mRNA abundance. However, there are several complexities built into the that can affect data interpretation. Most notably, selection of an appropriate internal control normalization essential expression In this study we investigated suitability seven commonly used genes [18S ribosomal RNA (18S), alpha tubulin (TUBA), beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), and polymerase II polypeptide B (POLR2B)] as potential quantitative references normalizing real-time generated in channel catfish physiology. Gene stability were evaluated among 15 tissues within physiologically-relevant response experimental manipulation (i.e. LHRH injection, fasting, acute stress). Expression candidate reference varied across all tissue types tested, indicating none could suitably serve cross comparisons. Experimentally altering physiological state fish differentially affected various depending on design type, with 18S unaffected by treatment examined. For example, expressed gene, GAPDH, opposed 18S, normalize hepatic growth hormone receptor during fasting resulted misinterpretation data. These results reveal importance providing comprehensive details gene validation when publishing results, manuscript serving basic guideline research.
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