Skip to main content
SpringerLink
Log in

Public-transcriptome-database-assisted selection and validation of reliable reference genes for qRT-PCR in rice

  • Research Paper
  • Published:
Science China Life Sciences Aims and scope Submit manuscript

Abstract

Accurate quantitative reverse transcription PCR (qRT-PCR) requires reliable reference genes whose expression does not vary in different tissues and developmental stages. However, few reliable reference genes are available for qRT-PCR in rice (Oryza sativa). Here, we established an effective strategy for identifying novel reference genes (NRGs) for reliable normalization of qRT-PCR data in various rice organs and developmental stages. We selected candidate NRGs using the Information Commons for Rice Database and confirmed their expression in Rice Expression Profile Database (RiceXPro) data. Genes with low variation (<2.5 cycle quantification) across tissues and developmental stages, and little fluctuation in expression in heatmaps from RiceXPro data were considered stable NRGs. To validate this strategy, we selected 11 candidate NRGs and calculated their expression stability in different spatio-temporal conditions using five programs, and compared these genes with five established reference genes (ERGs). Only one of the ERGs (UBQ5) was reliable and 10 of the candidate NRGs were more stable than the four remaining ERGs. Therefore, public transcriptomic databases are useful for identifying NRGs. We selected two NRGs, UFC1 (Homolog of UFM1-Conjugating Enzyme 1) and FhaB (Homolog of Adhesin FhaB) for qRT-PCR analysis in rice; their homologs might be suitable for other monocot plants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Akasaka, M., Taniguchi, Y., Oshima, M., Abe, K., Tabei, Y., and Tanaka, J. (2018). Development of transgenic male-sterile rice by using anther-specific promoters identified by comprehensive screening of the gene expression profile database ‘RiceXPro’. Breed Sci 68, 420–431.

    Article  CAS  Google Scholar 

  • Andersen, C.L., Jensen, J.L., and 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 Res 64, 5245–5250.

    Article  CAS  Google Scholar 

  • Arite, T., Iwata, H., Ohshima, K., Maekawa, M., Nakajima, M., Kojima, M., Sakakibara, H., and Kyozuka, J. (2007). DWARF10, an RMS1/MAX4/DAD1 ortholog, controls lateral bud outgrowth in rice. Plant J 51, 1019–1029.

    Article  CAS  Google Scholar 

  • Bustin, S.A., Benes, V., Nolan, T., and Pfaffl, M.W. (2005). Quantitative real-time RT-PCR—a perspective. J Mol Endocrinol 34, 597–601.

    Article  CAS  Google Scholar 

  • Chow, C.N., Zheng, H.Q., Wu, N.Y., Chien, C.H., Huang, H.D., Lee, T.Y., Chiang-Hsieh, Y. F., Hou, P. F., Yang, T. Y., and Chang, W. C. (2016). PlantPAN 2.0: an update of plant promoter analysis navigator for reconstructing transcriptional regulatory networks in plants. Nucleic Acids Res 44, D1154–D1160.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Gachon, C., Mingam, A., and Charrier, B. (2004). Real-time PCR: what relevance to plant studies? J Exp Bot 55, 1445–1454.

    Article  CAS  Google Scholar 

  • Jain, M. (2009). Genome-wide identification of novel internal control genes for normalization of gene expression during various stages of development in rice. Plant Sci 176, 702–706.

    Article  CAS  Google Scholar 

  • Jain, M., Nijhawan, A., Tyagi, A.K., and Khurana, J.P. (2006). Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun 345, 646–651.

    Article  CAS  Google Scholar 

  • Ji, Y., Tu, P., Wang, K., Gao, F., Yang, W., Zhu, Y., and Li, S. (2014). Defining reference genes for quantitative real-time PCR analysis of anther development in rice. Acta Biochim Biophys Sin 46, 305–312.

    Article  CAS  Google Scholar 

  • Kumar, D., Das, P.K., and Sarmah, B.K. (2018). Reference gene validation for normalization of RT-qPCR assay associated with germination and survival of rice under hypoxic condition. J Appl Genet 59, 419–430.

    Article  Google Scholar 

  • Kumar, V., Sharma, R., Trivedi, P.C., Vyas, G.K., and Khandelwal, V. (2011). Traditional and novel references towards systematic normalization of qRT-PCR data in plants. Aust J Crop Sci 5, 1455–1468.

    Google Scholar 

  • Li, J., Han, J., Hu, Y., and Yang, J. (2016). Selection of reference genes for quantitative real-time PCR during flower development in tree peony (Paeonia suffruticosa Andr.). Front Plant Sci 7.

  • Li, Q.F., Sun, S.S.M., Yuan, D.Y., Yu, H.X., Gu, M.H., and Liu, Q.Q. (2010). Validation of candidate reference genes for the accurate normalization of real-time quantitative RT-PCR Data in rice during seed development. Plant Mol Biol Rep 28, 49–57.

    Article  Google Scholar 

  • Livak, K.J., and Schmittgen, T.D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC T method. Methods 25, 402–408.

    Article  CAS  Google Scholar 

  • Ma, K., Han, J., Hao, Y., Yang, Z., Chen, J., Liu, Y.G., Zhu, Q., and Chen, L. (2019). An effective strategy to establish a male sterility mutant mini-library by CRISPR/Cas9-mediated knockout of anther-specific genes in rice. J Genet Genomics 46, 273–275.

    Article  Google Scholar 

  • Machado, R.D., Christoff, A.P., Loss-Morais, G., Margis-Pinheiro, M., Margis, R., and Körbes, A.P. (2015). Comprehensive selection of reference genes for quantitative gene expression analysis during seed development in Brassica napus. Plant Cell Rep 34, 1139–1149.

    Article  CAS  Google Scholar 

  • Narsai, R., Ivanova, A., Ng, S., and Whelan, J. (2010). Defining reference genes in Oryza sativa using organ, development, biotic and abiotic transcriptome datasets. BMC Plant Biol 10, 56.

    Article  Google Scholar 

  • Nonomura, K.I., Miyoshi, K., Eiguchi, M., Suzuki, T., Miyao, A., Hirochika, H., and Kurata, N. (2003). The MSP1 gene is necessary to restrict the number of cells entering into male and female sporogenesis and to initiate anther wall formation in rice. Plant Cell 15, 1728–1739.

    Article  CAS  Google Scholar 

  • Nonomura, K., Nakano, M., Eiguchi, M., Suzuki, T., and Kurata, N. (2006). PAIR2 is essential for homologous chromosome synapsis in rice meiosis I. J Cell Sci 119, 217–225.

    Article  CAS  Google Scholar 

  • Pfaffl, M.W., Tichopad, A., Prgomet, C., and Neuvians, T.P. (2004). Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—Excel-based tool using pair-wise correlations. Biotech Lett 26, 509–515.

    Article  CAS  Google Scholar 

  • Radonić, A., Thulke, S., Mackay, I.M., Landt, O., Siegert, W., and Nitsche, A. (2004). Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 313, 856–862.

    Article  Google Scholar 

  • Santos, F.I.C.D., Marini, N., Santos, R.S.D., Hoffman, B.S.F., Alves-Ferreira, M., and de Oliveira, A.C. (2018). Selection and testing of reference genes for accurate RT-qPCR in rice seedlings under iron toxicity. PLoS ONE 13, e0193418.

    Article  Google Scholar 

  • Sato, Y., Antonio, B.A., Namiki, N., Takehisa, H., Minami, H., Kamatsuki, K., Sugimoto, K., Shimizu, Y., Hirochika, H., and Nagamura, Y. (2010). RiceXPro: a platform for monitoring gene expression in japonica rice grown under natural field conditions. Nucleic Acids Res 39, D1141–D1148.

    Article  Google Scholar 

  • Sato, Y., Takehisa, H., Kamatsuki, K., Minami, H., Namiki, N., Ikawa, H., Ohyanagi, H., Sugimoto, K., Antonio, B.A., and Nagamura, Y. (2013). RiceXPro Version 3.0: expanding the informatics resource for rice transcriptome. Nucleic Acids Res 41, D1206–D1213.

    Article  CAS  Google Scholar 

  • Schmidt, G.W., and Delaney, S.K. (2010). Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress. Mol Genet Genomics 283, 233–241.

    Article  CAS  Google Scholar 

  • Silveira, E.D., Alves-Ferreira, M., Guimarães, L.A., da Silva, F.R., and 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 Biol 9, 84.

    Article  Google Scholar 

  • Silver, N., Best, S., Jiang, J., and Thein, S.L. (2006). Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol 7, 33.

    Article  Google Scholar 

  • Takehisa, H., Sato, Y., Igarashi, M., Abiko, T., Antonio, B.A., Kamatsuki, K., Minami, H., Namiki, N., Inukai, Y., Nakazono, M., et al. (2012). Genome-wide transcriptome dissection of the rice root system: implications for developmental and physiological functions. Plant J 69, 126–140.

    Article  CAS  Google Scholar 

  • Tang, X., Zhang, N., Si, H., and Calderón-Urrea, A. (2017). Selection and validation of reference genes for RT-qPCR analysis in potato under abiotic stress. Plant Methods 13, 85.

    Article  Google Scholar 

  • The IC4R, Project Consortium, Hao, L., Zhang, H., Zhang, Z., Hu, S., and Xue, Y. (2016). Information commons for rice (IC4R). Nucleic Acids Res 44, D1172–D1180.

    Article  Google Scholar 

  • Udvardi, M.K., Czechowski, T., and Scheible, W.R. (2008). Eleven golden rules of quantitative RT-PCR. Plant Cell 20, 1736–1737.

    Article  CAS  Google Scholar 

  • Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N., De Paepe, A., and Speleman, F. (2002). Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3, research0034.1.

    Article  Google Scholar 

  • Wang, D., Pei, K., Fu, Y., Sun, Z., Li, S., Liu, H., Tang, K., Han, B., and Tao, Y. (2007). Genome-wide analysis of the auxin response factors (ARF) gene family in rice (Oryza sativa). Gene 394, 13–24.

    Article  CAS  Google Scholar 

  • Wang, Z., Gerstein, M., and Snyder, M. (2009). RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10, 57–63.

    Article  CAS  Google Scholar 

  • Xia, L., Zou, D., Sang, J., Xu, X., Yin, H., Li, M., Wu, S., Hu, S., Hao, L., and Zhang, Z. (2017). Rice Expression Database (RED): an integrated RNA-Seq-derived gene expression database for rice. J Genet Genomics 44, 235–241.

    Article  Google Scholar 

  • Xie, F., Xiao, P., Chen, D., Xu, L., and Zhang, B. (2012). miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs. Plant Mol Biol 80, 75–84.

    Article  CAS  Google Scholar 

  • Xu, X., Hao, L., Zhu, J., Tang, B., Zhou, Q., Song, F., Chen, T., Zhang, S., Dong, L., Lan, L., et al. (2018). Database resources of the BIG data center in 2018. Nucleic Acids Res 46, D14–D20.

    Article  CAS  Google Scholar 

  • Zhang, D., Luo, X., and Zhu, L. (2011). Cytological analysis and genetic control of rice anther development. J Genet Genomics 38, 379–390.

    Article  CAS  Google Scholar 

  • Zhu, L., Yang, C., You, Y., Liang, W., Wang, N., Ma, F., and Li, C. (2019). Validation of reference genes for qRT-PCR analysis in peel and flesh of six apple cultivars (Malus domestica) at diverse stages of fruit development. Sci Horticult 244, 165–171.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from the National Key Research and Development Program of China (2016YFD0100601) and the National Natural Science Foundation of China (31772104, 31771739, 31600977).

Author information

Author notes

  1. Contributed equally to this work

Authors and Affiliations

  1. State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou, 510642, China

    Zhe Zhao, Zixu Zhang, Zhi Ding, Hengjun Meng, Rongxin Shen, Huiwu Tang, Yao-Guang Liu & Letian Chen

  2. Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, South China Agricultural University, Guangzhou, 510642, China

    Zhe Zhao, Zixu Zhang & Letian Chen

  3. Key Laboratory of Plant Functional Genomics and Biotechnology of Guangdong Provincial Higher Education Institutions, South China Agricultural University, Guangzhou, 510642, China

    Zhe Zhao, Zixu Zhang, Yao-Guang Liu & Letian Chen

  4. College of Life Sciences, South China Agricultural University, Guangzhou, 510642, China

    Zhe Zhao, Zixu Zhang, Zhi Ding, Hengjun Meng, Rongxin Shen, Huiwu Tang, Yao-Guang Liu & Letian Chen

Authors
  1. Zhe Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zixu Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhi Ding
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hengjun Meng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rongxin Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Huiwu Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yao-Guang Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Letian Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Letian Chen.

Ethics declarations

Compliance and ethics The author(s) declare that they have no conflict of interest.

Electronic Supplementary Material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhao, Z., Zhang, Z., Ding, Z. et al. Public-transcriptome-database-assisted selection and validation of reliable reference genes for qRT-PCR in rice. Sci. China Life Sci. 63, 92–101 (2020). https://doi.org/10.1007/s11427-019-1553-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11427-019-1553-5

Keywords

Search

Navigation