| Validation of housekeeping genes as internal controls for studying gene expression during Pacific oyster (Crassostrea gigas) development by quantitative real-time PCR | |
Du, Yishuai1,2; Zhang, Linlin1; Xu, Fei1; Huang, Baoyu1,2 ; Zhang, Guofan1; Li, Li1; Zhang, GF
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| 2013-03-01 | |
| 发表期刊 | FISH & SHELLFISH IMMUNOLOGY
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| ISSN | 1050-4648 |
| 卷号 | 34期号:3页码:939-945 |
| 文章类型 | Article |
| 摘要 | Hatchery-reared larvae of the Pacific oyster (Crassostrea gigas) often suffer from massive mortality induced by Ostreid herpesvirus 1 (OsHV-1) infection, indicating the importance of better understanding of oyster immune defense systems. The accuracy of measurements of gene expression levels based on quantitative real-time PCR assays relies on the use of housekeeping genes as internal controls; however, few studies have focused on the selection of such internal controls. In this study, we conducted a comprehensive investigation of internal control genes during oyster development in virus-infected and uninfected samples. Transcriptome data for 38 developmental stages were downloaded and the gene expression patterns were classified into 30 clusters. A total of 317 orthologs of classical housekeeping genes in the oyster genome were annotated. After combining the expression profiles and oyster housekeeping gene dataset, 14 candidate internal controls were selected for further investigation: Elongation factor-1 alpha (EF-1 alpha), 18S rRNA (18S), 28S rRNA (28S), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin (ACT), Ribosomal protein L7 (RL7), Ribosomal protein L27 (RL27), Ribosomal protein L36 (RL36), Ribosomal protein S18 (RS18), Heterogeneous nuclear ribonucleoprotein A2/B1 (RO21), Eukaryotic translation elongation factor 2 (EF2), Ubiquitin-conjugating enzyme E2D2 (UBCD1), S-phase kinase-associated protein 1 (SKP1) and Heterogeneous nuclear ribonucleoprotein Q (HNRPQ). RNA was extracted from oyster larvae infected with OsHV-1 (group A; GA), and OsHV-1 free larvae (group B; GB). The expression levels of the 14 candidate internal controls were studied in GA and GB larvae by real-time PCR. Their expression stabilities were further analyzed using the GeNorm program. RL7 and RS18 were the most stable genes in both OsHV-1 infected (GA) and uninfected (GB) larvae. These results suggest that RL7 and RS18 could be used as internal controls for studying gene expression in normal growing oyster larvae and in OsHV-1 infected larvae. These high quality internal controls will be a valuable resource in future studies of oyster larval mortality. (C) 2012 Elsevier Ltd. All rights reserved.; Hatchery-reared larvae of the Pacific oyster (Crassostrea gigas) often suffer from massive mortality induced by Ostreid herpesvirus 1 (OsHV-1) infection, indicating the importance of better understanding of oyster immune defense systems. The accuracy of measurements of gene expression levels based on quantitative real-time PCR assays relies on the use of housekeeping genes as internal controls; however, few studies have focused on the selection of such internal controls. In this study, we conducted a comprehensive investigation of internal control genes during oyster development in virus-infected and uninfected samples. Transcriptome data for 38 developmental stages were downloaded and the gene expression patterns were classified into 30 clusters. A total of 317 orthologs of classical housekeeping genes in the oyster genome were annotated. After combining the expression profiles and oyster housekeeping gene dataset, 14 candidate internal controls were selected for further investigation: Elongation factor-1 alpha (EF-1 alpha), 18S rRNA (18S), 28S rRNA (28S), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-actin (ACT), Ribosomal protein L7 (RL7), Ribosomal protein L27 (RL27), Ribosomal protein L36 (RL36), Ribosomal protein S18 (RS18), Heterogeneous nuclear ribonucleoprotein A2/B1 (RO21), Eukaryotic translation elongation factor 2 (EF2), Ubiquitin-conjugating enzyme E2D2 (UBCD1), S-phase kinase-associated protein 1 (SKP1) and Heterogeneous nuclear ribonucleoprotein Q (HNRPQ). RNA was extracted from oyster larvae infected with OsHV-1 (group A; GA), and OsHV-1 free larvae (group B; GB). The expression levels of the 14 candidate internal controls were studied in GA and GB larvae by real-time PCR. Their expression stabilities were further analyzed using the GeNorm program. RL7 and RS18 were the most stable genes in both OsHV-1 infected (GA) and uninfected (GB) larvae. These results suggest that RL7 and RS18 could be used as internal controls for studying gene expression in normal growing oyster larvae and in OsHV-1 infected larvae. These high quality internal controls will be a valuable resource in future studies of oyster larval mortality. (C) 2012 Elsevier Ltd. All rights reserved. |
| 关键词 | Internal Control Real-time Pcr Ostreid Herpesvirus 1 Development Larva |
| 学科领域 | Fisheries ; Immunology ; Marine & Freshwater Biology ; Veterinary Sciences |
| DOI | 10.1016/j.fsi.2012.12.007 |
| URL | 查看原文 |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS研究方向 | Fisheries ; Immunology ; Marine & Freshwater Biology ; Veterinary Sciences |
| WOS类目 | Fisheries ; Immunology ; Marine & Freshwater Biology ; Veterinary Sciences |
| WOS记录号 | WOS:000316523300023 |
| WOS关键词 | NORMAL HUMAN TISSUES ; RT-PCR ; QUANTIFICATION ; COMPENDIUM ; HEMOCYTES ; ISOFORMS ; HOMOLOG ; HYPOXIA ; STRESS ; FRANCE |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine |
| 引用统计 | |
| 文献类型 | 期刊论文 |
| 条目标识符 | http://ir.qdio.ac.cn/handle/337002/16729 |
| 专题 | 海洋生物技术研发中心 |
| 通讯作者 | Zhang, GF |
| 作者单位 | 1.Chinese Acad Sci, Inst Oceanol, Lab Marine Molluscan Genet & Breeding, Qingdao 266071, Shandong, Peoples R China 2.Univ Chinese Acad Sci, Beijing 10049, Peoples R China |
| 第一作者单位 | 中国科学院海洋研究所 |
| 推荐引用方式 GB/T 7714 | Du, Yishuai,Zhang, Linlin,Xu, Fei,et al. Validation of housekeeping genes as internal controls for studying gene expression during Pacific oyster (Crassostrea gigas) development by quantitative real-time PCR[J]. FISH & SHELLFISH IMMUNOLOGY,2013,34(3):939-945. |
| APA | Du, Yishuai.,Zhang, Linlin.,Xu, Fei.,Huang, Baoyu.,Zhang, Guofan.,...&Zhang, GF.(2013).Validation of housekeeping genes as internal controls for studying gene expression during Pacific oyster (Crassostrea gigas) development by quantitative real-time PCR.FISH & SHELLFISH IMMUNOLOGY,34(3),939-945. |
| MLA | Du, Yishuai,et al."Validation of housekeeping genes as internal controls for studying gene expression during Pacific oyster (Crassostrea gigas) development by quantitative real-time PCR".FISH & SHELLFISH IMMUNOLOGY 34.3(2013):939-945. |
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