Identification of reference genes for qRT-PCR analysis in Yesso scallop Patinopecten yessoensis.
Identification of reference genes for qRT-PCR analysis in Yesso scallop Patinopecten yessoensis.
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DOI：
10.1371/journal.pone.0075609
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Bao Z
中科院分区：
文献类型：
--
作者：
Feng L;Yu Q;Li X;Ning X;Wang J;Zou J;Zhang L;Wang S;Hu J;Hu X;Bao Z
双壳类约有30，000种现存物种，因其在生态系统、水产养殖和进化研究中的重要性而受到广泛关注。尽管实时定量逆转录PCR（qRT-PCR）在双壳类物种基因表达研究中的应用越来越多，但对于可靠和准确的qRT-PCR分析的参考基因选择的研究却很少。对于扇贝而言，缺乏可用于组织或胚胎/幼虫阶段的参考基因的系统评价，β-肌动蛋白（ACT）最常被用作未经验证的qRT-PCR参考基因。本研究从虾夷扇贝（Patinopecten yessoensis）的转录组数据中筛选出12个常用的候选参考基因，用于虾夷扇贝qRT-PCR参考基因的筛选。采用qRT-PCR方法检测了36份组织样品和15份胚胎/幼虫样品在正常生理条件下的表达情况，并采用geNorm、Normands和comparative RT-PCR Ct方法对这些基因的表达稳定性进行了评价。对于最稳定和最不稳定表达的基因，通过三种方法获得了相似的结果。结合3个程序的结果对12个基因进行综合排序，结果表明，DEAD-box RNA helicase（HELI）、ubiquitin（UBQ）和60 S ribosomal protein L16（RPL 16）是不同组织的最佳参考基因组合，而细胞色素B（CB）、细胞色素C（CC）、组蛋白H3.3（Histone H3.3）是不同胚胎/幼虫阶段的最佳参考基因组合3）和甘油醛-3-磷酸脱氢酶（GAPDH）被推荐用于qRT-PCR标准化。ACT是成体组织和胚胎/幼虫的最不稳定的基因之一。本研究首次系统分析了正常条件下扇贝qRT-PCR标准化参考基因的选择。所推荐的参比基因可为虾夷扇贝生物学过程相关基因的筛选提供参考，也可为其他扇贝或双壳类参比基因的筛选提供参考。
Bivalves comprise around 30,000 extant species and have received much attention for their importance in ecosystems, aquaculture and evolutionary studies. Despite the increasing application of real-time quantitative reverse transcription PCR (qRT-PCR) in gene expression studies on bivalve species, little research has been conducted on reference gene selection which is critical for reliable and accurate qRT-PCR analysis. For scallops, systematic evaluation of reference genes that can be used among tissues or embryo/larva stages is lacking, and β-actin (ACT) is most frequently used as qRT-PCR reference gene without validation. In this study, 12 commonly used candidate reference genes were selected from the transcriptome data of Yesso scallop ( Patinopecten yessoensis ) for suitable qRT-PCR reference genes identification. The expression of these genes in 36 tissue samples and 15 embryo/larva samples under normal physiological conditions was examined by qRT-PCR, and their expression stabilities were evaluated using three statistic algorithms, geNorm, NormFinder, and comparative ∆Ct method. Similar results were obtained by the three approaches for the most and the least stably expressed genes. Final comprehensive ranking for the 12 genes combing the results from the three programs showed that, for different tissues, DEAD-box RNA helicase (HELI), ubiquitin (UBQ), and 60S ribosomal protein L16 (RPL16) were the optimal reference genes combination, while for different embryo/larva stages, gene set containing Cytochrome B (CB), Cytochrome C (CC), Histone H3.3 (His3.3), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recommended for qRT-PCR normalization. ACT was among the least stable genes for both adult tissues and embryos/larvae. This work constitutes the first systematic analysis on reference genes selection for qRT-PCR normalization in scallop under normal conditions. The suitable reference genes we recommended will be useful for the identification of genes related to biological processes in Yesso scallop, and also in the reference gene selection for other scallop or bivalve species.