Quantification of rainbow trout (Oncorhynchus mykiss) estrogen receptor-alpha messenger RNA and its expression in the ovary during the reproductive cycle.

Abstract:

:This study developed a quantitative reverse transcription-polymerase chain reaction (RT-PCR) method to measure estrogen receptor-alpha (ERalpha) mRNA in the rainbow trout (Oncorhynchus mykiss). Using RT-PCR, and primers based on the known ERalpha DNA sequence in this species, cDNA sequences representing most of the protein coding region were obtained from ovary poly A(+) RNA. Using these DNA sequences as probes in Northern blot hybridizations confirmed that a single transcript of 4.2 kilobases in poly A(+) RNA could be detected in liver and ovary RNA. For the quantitative RT-PCR assay an internal standard RNA molecule was produced to control for inherent inter-tube differences in amplification efficiency and permit accurate quantification of ERalpha mRNAs. The quantitative RT-PCR assay proved to be highly specific for ERalpha mRNA with a detection limit of 6.9 fg, which corresponds to 273 fg ERalpha mRNA/microg total RNA. The quantitative RT-PCR assay was used to measure the levels of ERalpha mRNA in ovaries of rainbow trout at different stages of reproductive development. Ovarian ERalpha mRNA expression was found during two distinct periods of reproductive development, in pre-vitellogenic ovaries of fish with ovarian follicle diameters (OFDs) 1000 microm. ERalpha mRNA could not be detected in the ovaries of fish with OFDs >100 microm but 2000 microm.

journal_name

J Mol Endocrinol

authors

Nagler JJ,Krisfalusi M,Cyr DG

doi

10.1677/jme.0.0250243

subject

Has Abstract

pub_date

2000-10-01 00:00:00

pages

243-51

issue

2

eissn

0952-5041

issn

1479-6813

pii

JME00929

journal_volume

25

pub_type

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