Molecular characterization and mRNA expression of ribosomal protein L8 in Rana nigromaculata during development and under exposure to hormones
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Qinzhanfen
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DOI:
Received November 15, 2013,Revised February 23, 2014, Accepted February 24, 2014, Available online 2014-04-09
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Like Xenopus laevis, some species of the Rana genus are also used to study endocrine disrupting chemicals (EDCs). Although ribosomal protein L8 (rpl8) is the most used reference gene for analyzing gene expression by quantitative RT-PCR (qRT-PCR) in Rana, its suitability as the reference gene has never been validated in any species of the Rana genus. In this study, we characterized rpl8 cDNA in Rana nigromaculata, a promising native species in East Asia for assaying endocrine disrupting effects. We found that the rpl8 cDNA consisted of 919-bp and encoded 257 amino acids, exhibiting high identities of amino acid sequence with known rpl8 in other Rana species. Then, we examined the stability of mRNA expression during development. Compared with elongation factor 1 alpha 1 (EF1A1), another common housekeeping gene, neither stage-specific nor tissue-specific expression of rpl8 gene in all tissues examined (brain, liver, intestine, tail, testis and ovary) was found during R. nigromaculata development. Finally, we investigated rpl8 expression under exposure to hormones. No change in rpl8 mRNA expression was found under exposure to thyroid hormone (T4) and estrogen (estradiol), whereas expression of the corresponding biomarker genes was induced. Our results show that rpl8 is an appropriate reference gene for analyzing gene expression by qRT-PCR for assaying EDCs using R. nigromaculata, and might also provide a support for rpl8 as a reference gene in other Rana species due to the high conservation of rpl8 among the Rana genus.
