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首页> 外文期刊>Fisheries and Aquatic Sciences >Evaluation of reference genes for RT-qPCR study in abalone Haliotis discus hannai during heavy metal overload stress
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Evaluation of reference genes for RT-qPCR study in abalone Haliotis discus hannai during heavy metal overload stress

机译：重金属超负荷胁迫下鲍鱼鲍氏鲍氏菌RT-qPCR研究参考基因的评价

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Background: The evaluation of suitable reference genes as normalization controls is a prerequisite requirement for launching quantitative reverse transcription-PCR (RT-qPCR)-based expression study. In order to select the stable reference genes in abaloneHaliotis discus hannai tissues (gill and hepatopancreas) under heavy metal exposure conditions (Cu, Zn, and Cd), 12 potential candidate housekeeping genes were subjected to expression stability based on the comprehensive ranking while integrating four different statistical algorithms (geNorm, NormFinder, BestKeeper, and ACT method).Results: Expression stability in the gill subset was determined as RPL7>RPL8 > ACTB > RPL3 > PPIB > RPL7A > EF1A > RPL4 > GAPDH > RPL5 > UBE2 > B-TU. On the other hand, the ranking in the subset for hepatopancreas was RPL7> RPL3 > RPL8 > ACTB > RPL4 > EFIA > RPL5 > RPL7A > B-TU > UBE2 > PPIB > GAPDH. The pairwise variation assessed by the geNorm program indicates that two reference genes could be sufficient for accurate normalization in both gill and hepatopancreas subsets. Overall, both gill and hepatopancreas subsets recommended ribosomal protein genes (particularly RPL7) as stable references, whereas traditional housekeepers such as fi-tubulin (B-TU) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were ranked as unstable genes. The validation of reference gene selection was confirmed with the quantitative assay of MT transcripts.Conclusions: The present analysis showed the importance of validating reference genes with multiple algorithmic approaches to select genes that are truly stable. Our results indicate that expression stability of a given reference gene could not always have consensus across tissue types. The data from this study could be a good guide for the future design of RT-qPCR studies with respect to metal regulation/detoxification and other related physiologies in this abalone species.
机译：背景：评价合适的参考基因作为标准化对照是开展基于定量逆转录PCR（RT-qPCR）的表达研究的先决条件。为了在重金属暴露条件下（铜，锌和镉）在鲍鱼鲍鱼汉氏anna虫组织（g和肝胰腺）中选择稳定的参考基因，基于综合排名对12个潜在候选管家基因进行了表达稳定性分析，同时整合了四个结果：g子集中的表达稳定性确定为RPL7> RPL8> ACTB> RPL3> PPIB> RPL7A> EF1A> RPL4> GAPDH> RPL5> UBE2> B-恩另一方面，肝胰腺的子集中的排名是RPL7> RPL3> RPL8> ACTB> RPL4> EFIA> RPL5> RPL7A> B-TU> UBE2> PPIB> GAPDH。 geNorm程序评估的成对变异表明，两个参考基因可能足以在g和肝胰腺亚群中进行准确归一化。总体而言，g和肝胰腺亚群均推荐将核糖体蛋白基因（特别是RPL7）作为稳定的参考，而传统的管家如fi-tubulin（B-TU）和3-磷酸甘油醛脱氢酶（GAPDH）基因则被评为不稳定基因。结论：本分析表明，采用多种算法方法选择真正稳定的基因可以验证参考基因的重要性。我们的结果表明，给定参考基因的表达稳定性可能在不同组织类型之间不一定总是有共识的。这项研究的数据可能为将来设计此鲍鱼金属的调控/解毒及其他相关生理学方面的RT-qPCR研究提供很好的指导。

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