Espínola Sergio Martin, Ferreira Henrique Bunselmeyer, Zaha Arnaldo
Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
Departamento de Biologia Molecular e Biotecnologia, Instituto de Biociências, and Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
PLoS One. 2014 Jul 11;9(7):e102228. doi: 10.1371/journal.pone.0102228. eCollection 2014.
In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (βTUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1α, βACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1α and TBP were the most stable genes for both species. Interestingly, βACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1α as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1α was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1α reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus.
近年来，已发表了大量细粒棘球绦虫属的序列数据（包括基因组和转录组数据），从而便于分析在特定阶段表达或参与寄生虫发育的基因。为了进行合适的基因表达定量分析，强烈建议使用经过验证的参考基因。因此，本研究的目的是鉴定合适的参考基因，以便在诱导成虫前期早期发育时，对狭义细粒棘球绦虫（G1）和奥氏棘球绦虫中感兴趣的基因进行可靠的表达标准化。使用了来自狭义细粒棘球绦虫（G1）和奥氏棘球绦虫成虫前期的未经处理的原头蚴（PS）和经胃蛋白酶处理的原头蚴（PSP）。使用geNorm、Normfinder和RefFinder评估了11个候选参考基因（βTUB、NDUFV2、RPL13、TBP、CYP-1、RPII、EF-1α、βACT-1、GAPDH、ETIF4A-III和MAPK3）的基因表达稳定性。我们的qPCR数据与最近发表的RNA-seq数据显示出良好的相关性。关于表达稳定性，EF-1α和TBP是这两个物种中最稳定的基因。有趣的是，βACT-1（最常用的参考基因）以及GAPDH和ETIF4A-III（先前被鉴定为管家基因）在我们的实验条件下表现不稳定。我们建议在狭义细粒棘球绦虫和奥氏棘球绦虫的PS和PSP实验条件下进行涉及基因表达分析的研究时，使用EF-1α作为参考基因。为了证明其适用性，EF-1α被用作编码抗原B亚基的基因转录本相对定量中的标准化基因。相同的EF-1α参考基因可用于其他广义细粒棘球绦虫属物种的研究。本报告验证了扁形动物门绦虫纲物种的合适参考基因，从而为在其他具有重要流行病学意义的绦虫物种（如带绦虫属的物种）中进行进一步验证奠定了基础。
