LI Bing, CHEN Changsheng, XU Yan, JI Dehua, XIE Chaotian. Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis (Bangiales, Rhodophyta) by quantitative real-time PCR[J]. Acta Oceanologica Sinica, 2014, 33(9): 152-159. doi: 10.1007/s13131-014-0526-2
Citation: LI Bing, CHEN Changsheng, XU Yan, JI Dehua, XIE Chaotian. Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis (Bangiales, Rhodophyta) by quantitative real-time PCR[J]. Acta Oceanologica Sinica, 2014, 33(9): 152-159. doi: 10.1007/s13131-014-0526-2

Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis (Bangiales, Rhodophyta) by quantitative real-time PCR

doi: 10.1007/s13131-014-0526-2
  • Received Date: 2013-04-02
  • Rev Recd Date: 2013-12-18
  • Pyropia haitanensis is an economically important mariculture crop in China and has a high research value for several life phenomena, for example environmental tolerance. To explore the mechanisms underlying these characteristics, gene expression has been investigated at the whole transcriptome level. Gene expression studies using quantitative real-time PCR should start by selecting an appropriate internal control gene; therefore, the absolute expression abundance of six housekeeping genes (18S rRNA (18S), ubiquitin-conjugating enzyme (UBC), actin (ACT), β-tubulin (TUB), elongation factors 2 (EF2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) examined by the quantitative real-time PCR in samples corresponding to different strains, life-cycle stages and abiotic stress treatments. Their expression stabilities were assessed by the comparative cycle threshold (Ct) method and by two different software packages: geNorm and Norm-Finder. The most stable housekeeping gene is UBC and the least stable housekeeping is GADPH. Thus, it is proposed that the most appropriate internal control gene for expression analyses in P. haitanensis is UBC. The results pave the way for further gene expression analyses of different aspects of P. haitanensis biology including different strains, life-history stages and abiotic stress responses.
  • loading
  • Andersen C L, Jensen J L, Ørntoft T F. 2004. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Research, 64(15): 5245-5250
    Asamizu E, Nakajima M, Kitade Y, et al. 2003. Comparison of RNA expression profiles between the two generations of Porphyra yezoensis (Rhodophyta), based on expressed sequence tag frequency analysis. Journal of Phycology, 39(5): 923-930
    Blouin N A, Brodie J A, Grossma A C, et al. 2011. Porphyra: a marine crop shaped by stress. Trends Plant Sci, 16(1): 29-37
    Brunner A M, Yakovlev I A, Strauss S H. 2004. Validating internal controls for quantitative plant gene expression studies. BMC Plant Biology, 4: 14
    Bustin S A, Benes V, Garson J A, et al. 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55(4): 611-622
    Chang Ermei, Shi Shengqing, Liu Jianfeng, et al. 2012. Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae) using real-time PCR. PLoS ONE, 7(3): e33278
    Choi S, Hwang M S, Im S, et al. 2013. Transcriptome sequencing and comparative analysis of the gametophyte thalli of Pyropia tenera under normal and high temperature conditions. Journal of Applied Phycology, 25(4): 1237-1246
    Cole K, Conway E. 1975. Phenetic implications of structural features of the perennating phase in the life history of Porphyra and Bangia (Bangiophyceae, Rhodophyta). Phycologia, 14(4): 239-245
    Czechowski T, Stitt M, Altmann T, et al. 2005. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiology, 139(1): 5-17
    Dundas J, Ling M. 2012. Reference genes for measuring mRNA expression. Theory Biosci, 131(4): 215-223
    Faccioli P, Ciceri G P, Provero P, et al. 2007. A combined strategy of "in silico" transcriptome analysis and web search engine optimization allows an agile identification of reference genes suitable for normalization in gene expression studies. Plant Molecular Biology, 63(5): 679-688
    Fan Xiaolei, Fang Yongjun, Hu Songning, et al. 2007. Generation and analysis of 5318 expressed sequence tags from the filamentous sporophyte of Porphyra haitanensis (Rhodophyta). Journal of Phycology, 43(6): 1287-1294
    Flintoft L. 2011. Transcriptomics: measuring gene expression in nonmodel organisms. Nature Reviews Genetics, 12(11): 742
    Guénin S, Mauriat M, Pelloux J, et al. 2009. Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. Journal of Experimental Botany, 60(2): 487-493
    Guo R Y, Ki J S. 2012. Evaluation and validation of internal control genes for studying gene expression in the dinoflagellate Prorocentrum minimum using real-time PCR. European Journal of Protistology, 48(3): 199-206
    Hong S Y, Seo P J, Yang M S, et al. 2008. Exploring valid reference genes for gene expression studies in Brachypodium distachyon by realtime PCR. BMC Plant Biology, 8: 112
    Hu Ruibo, Fan Chengming, Li Hongyu, et al. 2009. Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. BMC Molecular Biology, 10: 93
    Iskandar H M, Simpson R S, Casu R E, et al. 2004. Comparison of reference genes for quantitative real-time polymerase chain reaction analysis of gene expression in sugarcane. Plant Molecular Biology Reporter, 22(4): 325-337 Jain M, Nijhawan A, Tyagi A K, et al. 2006. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochemical and Biophysical Research Communications, 345(2): 646-651
    Jian Bo, Liu Bin, Bi Yurong, et al. 2008. Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Molecular Biology, 9: 59
    Kakinuma M, Coury D A, Nakamoto C, et al. 2008. Molecular analysis of physiological responses to changes in nitrogen in a marine macroalga, Porphyra yezoensis (Rhodophyta). Cell Biology and Toxicology, 24(6): 629-639
    Kim B R, Nam H Y, Kim S U, et al. 2003. Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice. Biotechnology Letters, 25(21): 1869-1872
    Kim E, Park H S, Jung Y, et al. 2011. Identification of the high-temperature response genes from Porphyra seriata (Rhodophyta) expression sequence tags and enhancement of heat tolerance of Chlamydomonas (Chlorophyta) by expression of the Porphyra htr2 gene. Journal of Phycology, 47(4): 821-828 Le Bail A, Dittami S M, de Franco P O, et al. 2008. Normalisation genes for expression analyses in the brown alga model Ectocarpus siliculosus. BMC Molecular Biology, 9: 75
    Lee C, Kim J, Shin S G, et al. 2006. Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli. Journal of Biotechnology, 123(3): 273-280
    Libault M, Thibivilliers S, Bilgin D D, et al. 2008. Identification of four soybean reference genes for gene expression normalization. Plant Genome, 1(1): 44-54
    Mukai L S, Craigie J S, Brown R G. 1981. Chemical composition and structure of the cell walls of the conchocelis and thallus phases of Porphyra tenera (Rhodophyceae). Journal of Phycology, 17(2): 192-198
    Muers M. 2011. Gene expression: transcriptome to proteome and back to genome. Nat Rev Genet, 12(8): 518
    Nakamura Y, Sasaki N, Kobayashi M, et al. 2013. The first symbiontfree genome sequence of marine red alga, Susabi-nori (Pyropia yezoensis). PLoS ONE, 8(3): e57122
    Nicot N, Hausman J F, Hoffmann L, et al. 2005. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. Journal of Experimental Botany, 56(421): 2907-2914
    Nikaido I, Asamizu E, Nakajima M, et al. 2000. Generation of 10, 154 expressed sequence tags from a leafy gametophyte of a marine red alga, Porphyra yezoensis. DNA Research, 7(3): 223-227
    Niu Jianfeng, Gao Shenghan, Luo Yingfeng, et al. 2011. The analysis of the low coverage Porphyra yezoensis draft genome. Marine Sciences (in Chinese), 35(6): 76-81
    Paolacci A R, Tanzarella O A, Porceddu E, et al. 2009. Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Molecular Biology, 10: 11
    Pfaffl M W, Tichopad A, Prgomet C, et al. 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper-Excel-based tool using pair-wise correlations. Biotechnology Letters, 26(6): 509-515
    Radonić A, Thulke S, Mackay I M, et al. 2004. Guideline to reference gene selection for quantitative real-time PCR. Biochemical and Biophysical Research Communications, 313(4): 856-862
    Remans T, Smeets K, Opdenakker K, et al. 2008. Normalisation of real-time RT-PCR gene expression measurements in Arabidopsis thaliana exposed to increased metal concentrations. Planta, 227(6): 1343-1349
    Rosic N N, Pernice M, Rodriguez-Lanetty M, et al. 2011. Validation of housekeeping genes for gene expression studies in Symbiodinium exposed to thermal and light stress. Marine Biotechnology, 13(3): 355-365
    Sahoo D, Tang X R, Yarish C. 2002. Porphyra-the economic seaweed as a new experimental system. Current Science, 83(11): 1313-1316 Silver N, Best S, Jiang J, et al. 2006. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Molecular Biology, 7: 33
    Sutherland J E, Lindstrom S C, Nelson W A, et al. 2011. A new look at an ancient order: generic revision of the Bangiales (Rhodophyta). Journal of Phycology, 47(5): 1131-1151
    Tong Zhaoguo, Gao Zhihong, Wang Fei, et al. 2009. Selection of reliable reference genes for gene expression studies in peach using realtime PCR. BMC Molecular Biology, 10: 71
    Udvardi M K, Czechowski T, Scheible W R. 2008. Eleven golden rules of quantitative RT-PCR. Plant Cell, 20(7): 1736-1737
    Vandesompele J, De Preter K, Pattyn F, et al. 2002. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology, 3: RESEARCH0034
    Whelan J A, Russell N B, Whelan M A. 2003. A method for the absolute quantification of cDNA using real-time PCR. Journal of Immunological Methods, 278(1-2): 261-269
    Wu Xiaojie, Niu Jianfeng, Huang Aiyou, et al. 2012. Selection of internal control gene for expression studies in Porphyra haitanensis (Rhodophyta) at different life-history stages. Journal of Phycology, 48(4): 1040-1044
    Xie Chaotian, Chen Changsheng, Xu Yan, et al. 2010. Construction of a genetic linkage map for Porphyra haitanensis (Bangiales, Rhodophyta) based on sequence-related amplified polymorphism and simple sequence repeat markers. Journal of Phycology, 46(4): 780-787
    Yang Hui, Mao Yunxiang, Kong Fanna, et al. 2011. Profiling of the transcriptome of Porphyra yezoensis with Solexa sequencing technology. Chinese Science Bulletin, 56(20): 2119-2130
    Zhang Xuecheng, Qin Song, Ma Jiahai, et al. 2005. The Genetics of Marine Algae (in Chinese). Beijing: China Agriculture Press, 184-186
  • 加载中

Catalog

    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Article Metrics

    Article views (1944) PDF downloads(1180) Cited by()
    Proportional views
    Related

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return