Abstract
Quantitative real-time PCR (RT-qPCR) is a reliable method for assessing gene expression, provided that suitable reference genes are included to normalize the data. The stability of expression of eight potential reference genes, namely, tubulin (alpha-2,4 tubulin), actin, EF1α (elongation factor 1α), UBC (ubiquitin C), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), psaA (photosynthesis-related plastid gene representing photosystem I), PP2Acs (catalytic subunit of protein phosphatase 2A), and PGK (phosphoglycerate kinase), was assessed in chrysanthemum plants subjected to aphid infestation, heat stress or waterlogging stress using geNorm software. The widely used reference gene EF1α performed well for aphid infested plants but poorly for waterlogged ones. The catalytic subunit of protein phosphatase 2A (PP2Acs) was the best performing one during heat and waterlogging stress, but was the worst during aphid infestation. The commonly used reference gene actin was generally the least stable of the set. No single gene was suitable for normalization on its own. The choice of reference gene(s) is an important factor in gene expression studies based on RT-qPCR.
Similar content being viewed by others
References
Orsel, M., Krapp, A., & Daniel-Vedele, F. (2002). Analysis of the NRT2 nitrate transporter family in Arabidopsis. Structure and gene expression. Plant Physiology, 129, 886–896.
Bustin, S. (2000). Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology, 25, 169–193.
Bustin, S. (2002). Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. Journal of Molecular Endocrinology, 29, 23–39.
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 biology, 3(7).
Czechowski, T., Bari, R., Stitt, M., Scheible, W., & Udvardi, M. (2004). Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. The Plant Journal, 38, 366–379.
Jain, M., Nijhawan, A., Tyagi, A., & Khurana, J. (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, 646–651.
Brunner, A., Yakovlev, I., & Strauss, S. (2004). Validating internal controls for quantitative plant gene expression studies. BMC Plant Biology, 4, 14.
Paolacci, A., Tanzarella, O., Porceddu, E., & Ciaffi, M. (2009). Identification and validation of reference genes for quantitative RT-PCR normalization in wheat. BMC Molecular Biology, 10, 11.
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 Molecular Biology, 9, 59.
Deng, Y., Chen, S., Lu, A., Chen, F., Tang, F., Guan, Z., et al. (2010). Production and characterisation of the intergeneric hybrids between Dendranthema morifolium and Artemisia vulgaris exhibiting enhanced resistance to chrysanthemum aphid (Macrosiphoniellasanbourni). Planta, 231, 693–703.
Yin, D., Chen, S., Chen, F., Guan, Z., & Fang, W. (2009). Morphological and physiological responses of two chrysanthemum cultivars differing in their tolerance to waterlogging. Environmental and Experimental Botany, 67, 87–93.
Miao, H., Jiang, B., Chen, S., Zhang, S., Chen, F., Fang, W., et al. (2010). Isolation of a gibberellin 20-oxidase cDNA from and characterization of its expression in chrysanthemum. Plant Breeding, 129, 707–714.
Chen, S., Miao, H., Chen, F., Jiang, B., Lu, J., & Fang, W. (2009). Analysis of expressed sequence tags (ESTs) collected from the inflorescence of Chrysanthemum. Plant Molecular Biology Reporter, 27, 503–510.
Ramakers, C., Ruijter, J., Deprez, R., & Moorman, A. (2003). Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neuroscience Letters, 339, 62–66.
Livak, K., & Schmittgen, T. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2 −ΔΔCT method. Methods, 25, 402–408.
Nicot, N., Hausman, J., Hoffmann, L., & Evers, D. (2005). Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. Journal of Experimental Botany, 56, 2907–2914.
Løvdal, T., & Lillo, C. (2009). Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. Analytical Biochemistry, 387, 238–242.
Lin, Y., & Lai, Z. (2010). Reference gene selection for qPCR analysis during somatic embryogenesis in longan tree. Plant Science, 178, 359–365.
Fernandez, P., Di Rienzo, J. A., Moschen, S., Dosio, G. A. A., Aguirrezábal, L. A. N., Hopp, H. E., Paniego, N., & Heinz, R. A. (2010). Comparison of predictive methods and biological validation for qPCR reference genes in sunflower leaf senescence transcript analysis. Plant cell reports, 1–12 (2010). doi:10.1007/s00299-010-0944-3.
Xu, M., Zhang, B., Su, X., Zhang, S., & Huang, M. (2011). Reference gene selection for quantitative real-time polymerase chain reaction in Populus. Analytical Biochemistry, 408, 337–339.
Bezier, A., Lambert, B., & Baillieul, F. (2002). Study of defense-related gene expression in grapevine leaves and berries infected with Botrytis cinerea. European Journal of Plant Pathology, 108, 111–120.
Langer, K., Ache, P., Geiger, D., Stinzing, A., Arend, M., Wind, C., et al. (2002). Poplar potassium transporters capable of controlling K+ homeostasis and K+-dependent xylogenesis. The Plant Journal, 32, 997–1009.
Reid, K., Olsson, N., Schlosser, J., Peng, F., & Lund, S. (2006). An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biology, 6, 27.
Thomas, C., Meyer, D., Wolff, M., Himber, C., Alioua, M., & Steinmetz, A. (2003). Molecular characterization and spatial expression of the sunflower ABP1 gene. Plant Molecular Biology, 52, 1025–1036.
Tong, Z., Gao, Z., Wang, F., Zhou, J., & Zhang, Z. (2009). Selection of reliable reference genes for gene expression studies in peach using real-time PCR. BMC Molecular Biology, 10, 71.
Pérez, R., Tupac-Yupanqui, I., & Dunner, S. (2008). Evaluation of suitable reference genes for gene expression studies in bovine muscular tissue. BMC Molecular Biology, 9, 79.
Stürzenbaum, S., & Kille, P. (2001). Control genes in quantitative molecular biological techniques: the variability of invariance. Comparative Biochemistry and Physiology Part B, 130, 281–289.
Suzuki, T., Higgins, P., & Crawford, D. (2000). Control selection for RNA quantitation. Biotechniques, 29, 332–337.
McNulty, S., & Toscano, W. (1995). Transcriptional regulation of glyceraldehyde-3-phosphate dehydrogenase by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biochemical and Biophysical Research Communications, 212, 165–171.
Anderson, L., & Carol, A. (2005). Enzyme co-localization in the pea leaf cytosol: 3-P-glycerate kinase, glyceraldehyde-3-P dehydrogenase, triose-P isomerase and aldolase. Plant Science, 169, 620–628.
Andersen, C., Jensen, J., & Orntoft, T. (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, 5245–5250.
Martin, R. C., Hollenbeck, V. G., & Dombrowski, J. E. (2008). Evaluation of reference genes for quantitative RT-PCR in Lolium perenne. Crop Science, 48, 1881–1887.
Silveira, D., Alves-Ferreira, M., Guimar es, L. A., da Silva, F. R., & Carneiro, V. T. C. (2009). Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha. BMC Plant Biology, 9, 84.
Huis, R., Hawkins, S., & Neutelings, G. (2010). Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC Plant Biology, 10, 71.
Wan, H., Zhao, Z., Qian, C., Sui, Y., Malik, A. A., & Chen, J. (2010). Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Analytical Biochemistry, 399, 257–261.
Thellin, O., Zorzi, W., Lakaye, B., De Borman, B., Coumans, B., Hennen, G., et al. (1999). Housekeeping genes as internal standards: use and limits. Journal of Biotechnology, 75, 291–295.
Acknowledgments
This study is supported by the National Natural Science Foundation of China (Grant No. 30872064, 31071820, 31071825), the Program for Hi-Tech Research, Jiangsu, China, Grant (No. BE2008307, BE2009317, BE2010303), and the Fundamental Research Funds for the Central Universities (KYJ 200907).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Gu, C., Chen, S., Liu, Z. et al. Reference Gene Selection for Quantitative Real-Time PCR in Chrysanthemum Subjected to Biotic and Abiotic Stress. Mol Biotechnol 49, 192–197 (2011). https://doi.org/10.1007/s12033-011-9394-6
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12033-011-9394-6