Identification of normalization factors for quantitative realtime RT-PCR analysis of gene expression in Pacific abalone Haliotis discus hannai  

作　　者：邱礽 孙铂光 房沙沙 孙黎 刘晓 

机构地区：[1]Institute of Oceanology,Chinese Academy of Sciences [2]Graduate University of Chinese Academy of Sciences

出　　处：《Chinese Journal of Oceanology and Limnology》2013年第2期421-430,共10页中国海洋湖沼学报（英文版）

基　　金：Supported by the Knowledge Innovation Program of Chinese Academy of Sciences(No.KSCX2-EW-G-12B);the Knowledge Innovation Program of the Chinese Academy of Sciences(No.KZCX2-EW-Q213);the National High Technology Research and Development Program of China (863 Program)(No.2012AA10A412)

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 Fibrio 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-l-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 

分 类 号：S917.4[农业科学—水产科学]