The Establishment of a Quantitative RT-PCR Assay for Estrogen Receptor mRNA in Japanese Eel, Anguilla japonica(Endocrinology)

概要

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A sensitive quantitation system using reverse transcription-polymerase chain reaction (RT-PCR) was developed to measure the low estrogen receptor (ER) mRNA levels in Japanese eel (Anguilla japonica). Two types of cytoskeletal actins, β- and γ-actins, were distinguished in Japanese eel and used as the internal control for exact quantitation. Actin and ER primers of this study annealed to different exons, allowing for the skipping of Dnase treatment. Accordingly, ER and actin RT-PCR products showed a single band and were amplified with the same efficiency during PCR. The ER mRNA amount was calculated as a relative value, normalized over actin (β and γ) mRNA. The results thus obtained by RT-PCR agreed with the results from Northern blot analysis of liver from pre-vitellogenic and hormone-treated early vitellogenic eel. Using this system, the ER mRNA levels were further measured in coelomic epithelium, pituitary, brain and ovary. In the liver, ER mRNA levels of the early vitellogenic eel increased by about 2.7 folds compared to those of the immature eel. In contrast, changes in levels of ER transcripts were not observed in other tissues. This system can be used to detect relative ER mRNA levels around 100-fold lower than those of actin mRNA in all tissues in which it has been difficult to measure ER mRNA by Northern blot analysis.
社団法人日本動物学会の論文
1998-12-15