Validation of housekeeping genes as internal controls for studying gene expression during Pacific oyster (Crassostrea gigas) development by quantitative real-time PCR

	Validation of housekeeping genes as internal controls for studying gene expression during Pacific oyster (Crassostrea gigas) development by quantitative real-time PCR
	Du, Yishuai 1,2; Zhang, Linlin 1; Xu, Fei 1; Huang, Baoyu1,2 ; Zhang, Guofan 1; Li, Li 1
刊名	FISH & SHELLFISH IMMUNOLOGY
	2013-03-01
卷号	34 期号:3 页码:939-945
关键词	Internal control Real-time PCR Ostreid herpesvirus 1 Development Larva
ISSN号	1050-4648
通讯作者	Zhang, GF
中文摘要	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.
英文摘要	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.
学科主题	Fisheries ; Immunology ; Marine & Freshwater Biology ; Veterinary Sciences
WOS标题词	Science & Technology ; Life Sciences & Biomedicine
类目[WOS]	Fisheries ; Immunology ; Marine & Freshwater Biology ; Veterinary Sciences
研究领域[WOS]	Fisheries ; Immunology ; Marine & Freshwater Biology ; Veterinary Sciences
关键词[WOS]	NORMAL HUMAN TISSUES ; RT-PCR ; QUANTIFICATION ; COMPENDIUM ; HEMOCYTES ; ISOFORMS ; HOMOLOG ; HYPOXIA ; STRESS ; FRANCE
收录类别	SCI
原文出处	10.1016/j.fsi.2012.12.007
语种	英语
WOS记录号	WOS:000316523300023
公开日期	2014-07-17
内容类型	期刊论文
源URL	[http://ir.qdio.ac.cn/handle/337002/16729]
专题	海洋研究所_海洋生物技术研发中心
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
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,&Li, Li.(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.