Institutional Repository of Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences
| Identification of normalization factors for quantitative real-time RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai | |
Qiu Reng1,2; Sun Boguang1 ; Fang Shasha1,2; Sun Li1; Liu Xiao1; Sun, L
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| 2013-03-01 | |
| 发表期刊 | CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY
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| ISSN | 0254-4059 |
| 卷号 | 31期号:2页码:421-430 |
| 文章类型 | Article |
| 摘要 | Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Vibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues (digestive glands, foot muscle, gill, hemocyte, and mantle) were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-1-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points.; Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is widely used in studies of gene expression. In most of these studies, housekeeping genes are used as internal references without validation. To identify appropriate reference genes for qRT-PCR in Pacific abalone Haliotis discus hannai, we examined the transcription stability of six housekeeping genes in abalone tissues in the presence and absence of bacterial infection. For this purpose, abalone were infected with the bacterial pathogen Vibrio anguillarum for 12 h and 48 h. The mRNA levels of the housekeeping genes in five tissues (digestive glands, foot muscle, gill, hemocyte, and mantle) were determined by qRT-PCR. The PCR data was subsequently analyzed with the geNorm and NormFinder algorithms. The results show that in the absence of bacterial infection, elongation factor-1-alpha and beta-actin were the most stably expressed genes in all tissues, and thus are suitable as cross-tissue type normalization factors. However, we did not identify any universal reference genes post infection because the most stable genes varied between tissue types. Furthermore, for most tissues, the optimal reference genes identified by both algorithms at 12 h and 48 h post-infection differed. These results indicate that bacterial infection induced significant changes in the expression of abalone housekeeping genes in a manner that is dependent on tissue type and duration of infection. As a result, different normalization factors must be used for different tissues at different infection points. |
| 关键词 | Haliotis Discus Hannai Housekeeping Gene Normalization Factor Quantitative Real Time Rt-pcr Reference Gene |
| 学科领域 | Marine & Freshwater Biology ; Oceanography |
| DOI | 10.1007/s00343-013-2221-0 |
| URL | 查看原文 |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS研究方向 | Marine & Freshwater Biology ; Oceanography |
| WOS类目 | Limnology ; Oceanography |
| WOS记录号 | WOS:000316018300019 |
| WOS关键词 | POLYMERASE CHAIN-REACTION ; HOUSEKEEPING GENES ; DIVERSICOLOR-SUPERTEXTA ; VIBRIO-PARAHAEMOLYTICUS ; INTERNAL CONTROL ; RIBOSOMAL-RNA ; SELECTION ; VALIDATION ; ACTIN ; CHINA |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine ; Physical Sciences |
| 引用统计 | |
| 文献类型 | 期刊论文 |
| 条目标识符 | http://ir.qdio.ac.cn/handle/337002/16590 |
| 专题 | 实验海洋生物学重点实验室 |
| 通讯作者 | Sun, L |
| 作者单位 | 1.Chinese Acad Sci, Inst Oceanol, Qingdao 266071, Peoples R China 2.Chinese Acad Sci, Grad Univ, Beijing 100049, Peoples R China |
| 推荐引用方式 GB/T 7714 | Qiu Reng,Sun Boguang,Fang Shasha,et al. Identification of normalization factors for quantitative real-time RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai[J]. CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY,2013,31(2):421-430. |
| APA | Qiu Reng,Sun Boguang,Fang Shasha,Sun Li,Liu Xiao,&Sun, L.(2013).Identification of normalization factors for quantitative real-time RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai.CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY,31(2),421-430. |
| MLA | Qiu Reng,et al."Identification of normalization factors for quantitative real-time RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai".CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY 31.2(2013):421-430. |
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