Skip to main content

Selection of reference genes for gene expression normalization in Pyropia yezoensis using quantitative real-time PCR

Abstract

Quantitative real-time PCR (qRT-PCR) is frequently used for gene expression analysis. The selection of reference genes is required for normalization of the variation to avoid misinterpretation of experimental results and erroneous analyses. Pyropia yezoensis, growing in the intertidal zone, is an economically important seaweed. Although this intertidal seaweed has been an experimental system for understanding stress tolerance and developmental mechanisms, reference genes suitable for the normalization of qRT-PCR data have not previously been identified. In this study, expression stability of six candidate reference genes, including ACT, eIF4A, EF1α, GAPDH, TUA, and UBQ (traditional housekeeping genes), has been validated in a diverse set of samples representing different developmental stages and stress conditions of P. yezoensis. Three qRT-PCR analysis methods, geNorm, NormFinder, and BestKeeper, were evaluated systematically. The results indicated that ACT3, eIF4A, and EF1α were the optimal reference genes for P. yezoensis under stress conditions; UBQ, EF1α, and eIF4A were suitable for studying the expression of genes related to P. yezoensis development. TUA showed the lowest expression stability both under stress conditions and over developmental stages. Our results have provided a reference gene application guideline for P. yezoensis gene expression characterization using the qRT-PCR system.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3

References

  • Andersen CL, Jensen JL, Orntoft TF (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 Res 64:5245–5250

    Article  CAS  PubMed  Google Scholar 

  • Artico S, Nardeli SM, Brilhante O, Grossi-de-Sa MF, Alves-Ferreira M (2010) Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data. BMC Plant Biol 10:49

    Article  PubMed Central  PubMed  Google Scholar 

  • Barsalobres-Cavallari CF, Severino FE, Maluf MP, Maia IG (2009) Identification of suitable internal control genes for expression studies in Coffea arabica under different experimental conditions. BMC Mol Biol 10:1

    Article  PubMed Central  PubMed  Google Scholar 

  • Blouin NA, Brodie JA, Grossman AC, Xu P, Brawley SH (2010) Porphyra: a marine crop shaped by stress. Trends Plant Sci 16:29–37

    Article  PubMed  Google Scholar 

  • Butte AJ, Dzau VJ, Glueck SB (2001) Further defining housekeeping, or “maintenance”, genes focus on “a compendium of gene expression in normal human tissues”. Physiol Genomics 7:95–96

    CAS  PubMed  Google Scholar 

  • Chandna R, Augustine R, Bisht NC (2012) Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR. PLoS One 7(5):e36918

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Chang E, Shi S, Liu J, Cheng T, Xue L, Yang X, Yang W, Lan Q, Jiang Z (2012) Selection of reference genes for quantitative gene expression studies in Platycladus orientalis (Cupressaceae) Using real-time PCR. PLoS One 7(3):e33278

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Chen X, Truksa M, Shah S, Weselake RJ (2010) A survey of quantitative real-time polymerase chain reaction internal reference genes for expression studies in Brassica napus. Anal Biochem 405:138–140

    Article  CAS  PubMed  Google Scholar 

  • Chen L, Zhong HY, Kuang JF, Li JG, Lu WJ, Chen JY (2011) Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions. Planta 234:377–390

    Article  CAS  PubMed  Google Scholar 

  • Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Davison IR, Pearson GA (1996) Stress tolerance in intertidal seaweeds. J Phycol 32:197–211

    Article  Google Scholar 

  • de Oliveira LA, Breton MC, Bastolla FM, Camargo Sda S, Margis R, Frazzon J, Pasquali G (2012) Reference genes for the normalization of gene expression in Eucalyptus species. Plant Cell Physiol 53:405–422

    Article  PubMed  Google Scholar 

  • Die JV, Roman B, Nadal S, Gonzalez-Verdejo CI (2010) Evaluation of candidate reference genes for expression studies in Pisum sativum under different experimental conditions. Planta 232:145–153

    Article  CAS  PubMed  Google Scholar 

  • Dutcher SK (2001) The tubulin fraternity: alpha to eta. Curr Opin Cell Biol 13:49–54

    Article  CAS  PubMed  Google Scholar 

  • Gu C, Chen S, Liu Z, Shan H, Luo H, Guan Z, Chen F (2011) Reference gene selection for quantitative real-time PCR in Chrysanthemum subjected to biotic and abiotic stress. Mol Biotechnol 49:192–197

    Article  CAS  PubMed  Google Scholar 

  • Gutierrez L, Mauriat M, Guenin S, Pelloux J, Lefebvre JF, Louvet R, Rusterucci C, Moritz T, Guerineau F, Bellini C, Van Wuytswinkel O (2008) The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants. Plant Biotechnol J 6:609–618

    Article  CAS  PubMed  Google Scholar 

  • Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8(2):R19

    Article  PubMed Central  PubMed  Google Scholar 

  • Hong SY, Seo PJ, Yang MS, Xiang F, Park CM (2008) Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR. BMC Plant Biol 8:112

    Article  PubMed Central  PubMed  Google Scholar 

  • Hu R, Fan C, Li H, Zhang Q, Fu YF (2009) Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR. BMC Mol Biol 10:93

    Article  PubMed Central  PubMed  Google Scholar 

  • Jarosova J, Kundu JK (2010) Validation of reference genes as internal control for studying viral infections in cereals by quantitative real-time RT-PCR. BMC Plant Biol 10:146

    Article  PubMed Central  PubMed  Google Scholar 

  • Jian B, Liu B, Bi Y, Hou W, Wu C, Han T (2008) Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Mol Biol 9:59

    Article  PubMed Central  PubMed  Google Scholar 

  • Le Bail A, Dittami SM, de Franco PO, Rousvoal S, Cock MJ, Tonon T, Charrier B (2008) Normalisation genes for expression analyses in the brown alga model Ectocarpus siliculosus. BMC Mol Biol 9:75

    Article  PubMed Central  PubMed  Google Scholar 

  • Li L, Yan Y, Xu H, Qu T, Wang B (2011) Selection of reference genes for gene expression studies in ultraviolet B-irradiated human skin fibroblasts using quantitative real-time PCR. BMC Mol Biol 12:8

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Maroufi A, Van Bockstaele E, De Loose M (2010) Validation of reference genes for gene expression analysis in chicory (Cichorium intybus) using quantitative real-time PCR. BMC Mol Biol 11:15

    Article  PubMed Central  PubMed  Google Scholar 

  • Mehdi Khanlou K, Van Bockstaele E (2012) A critique of widely used normalization software tools and an alternative method to identify reliable reference genes in red clover (Trifolium pratense L.). Planta 236:1381–1393

    Article  CAS  PubMed  Google Scholar 

  • Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot 56:2907–2914

    Article  CAS  PubMed  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Provasoli L (1968) Media and prospects for the cultivation of marine algae. In: Watanabe A, Hattori A (eds) Cultures and Collections of Algae. Japanese Society Plant Physiology, Hakone, pp 63–75

    Google Scholar 

  • Reid KE, Olsson N, Schlosser J, Peng F, Lund ST (2006) An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol 6:27

    Article  PubMed Central  PubMed  Google Scholar 

  • Sahoo D, Tang X, Yarish C (2002) Porphyra—the economic seaweed as a new experimental system. Curr Sci 83:1313–1316

    Google Scholar 

  • Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108

    Article  CAS  PubMed  Google Scholar 

  • Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, Speleman F (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3(7):RESEARCH0034

    Article  PubMed Central  PubMed  Google Scholar 

  • Wan H, Zhao Z, Qian C, Sui Y, Malik AA, Chen J (2010) Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Anal Biochem 399:257–261

    Article  CAS  PubMed  Google Scholar 

  • Yang H, Mao Y, Kong F, Yang G, Ma F, Wang L (2011) Profiling of the transcriptome of Porphyra yezoensis with Solexa sequencing technology. Chinese Sci Bull 56(20):2119–2130

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National High Technology Research and Development Program of China (Grant No. 2012AA10A401, No. 2012AA10A406, and No. 2012AA100815), National Natural Science Foundation of China (Grant No. 31372517), the Public Science and Technology Research Funds Projects of Agriculture (Grant No. 200903030), and National Infrastructure of Fishery Germplasm Resources.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Yunxiang Mao.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kong, F., Cao, M., Sun, P. et al. Selection of reference genes for gene expression normalization in Pyropia yezoensis using quantitative real-time PCR. J Appl Phycol 27, 1003–1010 (2015). https://doi.org/10.1007/s10811-014-0359-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10811-014-0359-6

Keywords

  • BestKeeper
  • GeNorm
  • NormFinder
  • Quantitative real-time PCR
  • Reference gene
  • Pyropia yezoensis
  • Rhodophyta