Reference genes for quantitative real-time PCR analysis and quantitative expression of P5CS in Agropyron mongolicum under drought stress

Reference genes, stably expressing in different tissues and cells, are commonly used as the references in expression analysis. Selecting the optimum reference gene is crucial to the success of experiments. In this study, the expression stabilities of nine common reference genes, including ACT2, 18 S r RNA, APRT, EF-1α, RNA POL II, TUBα, TUBβ, GAPDH and TLF of Agropyron mongolicum, were studied under drought condition. Among them, 18 S r RNA was found to be the most optimum reference gene under drought stress by the analyzing of ge Norm and Norm Finder software. Quantitative expression levels of P5 CS using 18 S r RNA as the reference gene, and proline contents under drought stress in A. mongolicum were further operated, and we found the expression level of P5 CS gene and proline content had a significantly positive relationship（R^2=0.7763, P〈0.05）. This study established and validated 18 S r RNA as the reference genes in A. mongolicum under drought stress, providing a powerful tool for the quantitative expression analysis of drought genes in A. mongolicum.

Molecular characterization and m RNA expression of ribosomal protein L8 in Rana nigromaculata during development and under exposure to hormones

Like Xenopus laeuis, some species of the Rang genus are also used to study endocrine disrupting chemicals （EDCs）. Although ribosomal protein L8 （rp18） is the most-used reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction in Rang, its suitability as the reference gene has never been validated in any species of the Rana genus. We characterized rp18 cDNA in Rana nigromaculata, a promising native species in East Asia for assaying endocrine disrupting effects. We found that the rp18 cDNA consisted of 919 bp and encoded 257 amino acids, exhibiting high identities of amino acid sequence with known rp18 in other Rana species. Then, we examined the stability of mRNA expression during development. Compared with elongation factor 1 alpha 1, another common housekeeping gene, neither stage-specific nor tissue-specific expression of the rp18 gene was found in all tissues examined （brain, liver, intestine, tail, testis and ovary） during R. nigromaculata development. Finally, we investigated rp18 expression under exposure to hormones. No change in rp18 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 rp18 is an appropriate reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction for assaying EDCs using R. nigromaculata, and might also provide support for using rp18 as a reference gene in other Rang species due to the high conservation of rp18 among the Rana genus.