Skip to main content

Suitable internal control genes for qRT-PCR normalization in cotton fiber development and somatic embryogenesis

Chinese Science Bulletin volume 52, pages 3110–3117 (2007)Cite this article

Abstract

The mechanisms of cotton fiber development and somatic embryogenesis have been explored systematically with microarray and suppression subtractive hybridization. Real-time RT-PCR provides the simultaneous measurement of gene expression in many different samples, with which the data from microarray or others can be confirmed in detail. To achieve accurate and reliable gene expression results, normalization of real-time PCR data against one or several internal control genes is required, which should not fluctuate in different tissues during various stages of development. We assessed the gene expression of 7 frequently used housekeeping genes, including 18S rRNA, Histone3, UBQ7, Actin, Cyclophilin, Gbpolyubiquitin-1 and Gbpolyubiquitin-2, in a diverse set of 21 cotton samples. For fiber developmental series the expression of all housekeeping genes had the same down tendency after 17 DPA. But the expression of the AGP gene (arabinogalactan protein) that has high expression level at the later fiber development stage was up-regulated from 15 to 27 DPA. So the relative absolute quantification should be an efficient and convenient method for the fiber developmental series. The expression of nonfiber tissues series varied not so much against the fiber developmental series. And three best control genes Histone3, UBQ7 and Gbpolyubiquitin-1 have to be used in a combinated way to get better normalization.

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

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

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

References

  1. Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol, 2002, 3: RESEARCH0034

  2. Thellin O, Zorzi W, Lakaye B, et al. Housekeeping genes as internal standards: Use and limits. J Biotechnol, 1999, 75: 291–295

    Article  Google Scholar 

  3. Lee P D, Sladek R, Greenwood C M, et al. Control genes and variability: Absence of ubiquitous reference transcripts in diverse mammalian expression studies. Genome Res, 2002, 12: 292–297

    Article  Google Scholar 

  4. Czechowski T, Stitt M, Altmann T, et al. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol, 2005, 139: 5–17

    Article  Google Scholar 

  5. Pfaffl M W, Tichopad A, Prgomet C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper—Excel-based tool using pair-wise correlations. Biotechnol Lett, 2004, 26: 509–515

    Article  Google Scholar 

  6. Goidin D, Mamessier A, Staquet M J, et al. Ribosomal 18S rRNA prevails over glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations. Anal Biochem, 2001, 295: 17–21

    Article  Google Scholar 

  7. Andersen C L, Jensen J L, Orntoft T F. 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, 2004, 64: 5245–5250

    Article  Google Scholar 

  8. Brunner A M, Yakovlev I A, Strauss S H. Validating internal controls for quantitative plant gene expression studies. BMC Plant Biol, 2004, 4: 14

    Article  Google Scholar 

  9. Dheda K, Huggett J F, Bustin S A, et al. Validation of housekeeping genes for normalizing RNA expression in real-time PCR. Biotechniques, 2004, 37: 112–114

    Google Scholar 

  10. Radonic A, Thulke S, Mackay I M, et al. Guideline to reference gene selection for quantitative realtime PCR. Biochem Biophys Res Commun, 2004, 313: 856–862

    Article  Google Scholar 

  11. Jain M, Nijhawan A, Tyagi A K, et al. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun, 2006, 345: 646–651

    Article  Google Scholar 

  12. Arpat A B, Waugh M, Sullivan J P, et al. Functional genomics of cell elongation in developing cotton fibers. Plant Mol Biol, 2004, 54: 911–929

    Article  Google Scholar 

  13. Li X B, Fan X P, Wang X L, et al. The cotton ACTIN1 gene is functionally expressed in fibers and participates in fiber elongation. Plant Cell, 2005, 17: 859–875

    Article  Google Scholar 

  14. Lee J J, Hassan O S, Gao W, et al. Developmental and gene expression analyses of a cotton naked seed mutant. Planta, 2006, 223: 418–432

    Article  Google Scholar 

  15. Shi Y H, Zhu S W, Mao X Z, et al. Transcriptome profiling, molecular biological, and physiological studies reveal a major role for ethylene in cotton fiber cell elongation. Plant Cell 2006, 18: 651–664

    Article  Google Scholar 

  16. Samuel Yang S, Cheung F, Lee J J, et al. Accumulation of genome-specific transcripts, transcription factors and phytohormonal regulators during early stages of fiber cell development in allotetraploid cotton. Plant J, 2006, 47: 761–775

    Article  Google Scholar 

  17. Zeng F, Zhang X, Zhu L, et al. Isolation and characterization of Genes associated to cotton somatic embryogenesis by suppression subtractive hybridization and macroarray. Plant Mol Biol, 2006, 60: 167–183

    Article  Google Scholar 

  18. Wu J H, Zhang X L, Nie Y C, et al. Factors affecting somatic embryogenesis and plant regeneration from a range of recalcitrant genotypes of Chinese cotton (Gossypium hirsutum L.). In Vitro Cell Del Biol Plant, 2004, 40: 371–375

    Article  Google Scholar 

  19. Zhu L F, Tu L L, Zeng F C, et al. An improved simple protocol for isolation of high quality RNA from Gossypium spp. suitable for cDNA library construction. Acta Agronomica Sinica, 2005, 31: 1657–1659

    Google Scholar 

  20. Sun Y, Zhang X, Huang C, et al. Factors influencing in vitro regeneration from protoplasts of wild cotton (G. klotzschianum A) and RAPD analysis of regenerated plantlets. Plant Growth Regul, 2005, 46: 79–86

    Article  Google Scholar 

  21. Liu D, Zhang X, Tu L, et al. Isolation by suppression-subtractive hybridization of genes preferentially expressed during early and late fiber development stages in cotton. Mol Biol (Mosk), 2006, 40: 825–834

    Google Scholar 

  22. Kim B R, Nam H Y, Kim S U, et al. Normalization of reverse transcription quantitative-PCR with housekeeping genes in rice. Biotechnol Lett, 2003, 25: 1869–1872

    Article  Google Scholar 

  23. Nicot N, Hausman J F, Hoffmann L, et al. Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot, 2005, 56: 2907–2914

    Article  Google Scholar 

  24. Goncalves S, Cairney J, Maroco J, et al. Evaluation of control transcripts in real-time RT-PCR expression analysis during maritime pine embryogenesis. Planta, 2005, 222: 556–563

    Article  Google Scholar 

  25. Reid K E, Olsson N, Schlosser J, et al. An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol, 2006, 14: 27

    Article  Google Scholar 

  26. Laney J D, Hochstrasser M. Substrate targeting in the ubiquitin system. Cell, 1999, 97: 427–430

    Article  Google Scholar 

  27. Callis J, Raasch J A, Vierstra R D. Ubiquitin extension proteins of Arabidopsis thaliana. J Bio Chem, 1999, 265: 12486–12493

    Google Scholar 

  28. Ciechanover A. The ubiquitin-proteasome pathway: On protein death and cell life. EMBO J, 1998, 17: 7151–7160

    Article  Google Scholar 

  29. Hochstrasser M. Evolution and function of ubiquitin-like protein-conjugation systems. Nat Cell Biol, 2000, 2: E153–E157

    Article  Google Scholar 

  30. Basra A, Malik C P. Development of the cotton fiber. Int Rev Cytol, 1984, 89: 65–113

    Article  Google Scholar 

  31. Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the \(2^{ - \Delta \Delta C_T } \) method. Methods, 2001, 25: 402–408

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, China

    Tu LiLi, Zhang XianLong, Liu DiQiu, Jin ShuangXia, Cao JingLin, Zhu LongFu, Deng FengLin, Tan JiaFu & Zhang CunBin

Authors
  1. Tu LiLi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhang XianLong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Liu DiQiu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jin ShuangXia
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Cao JingLin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zhu LongFu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Deng FengLin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Tan JiaFu
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Zhang CunBin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Zhang XianLong.

Additional information

Supported by the National High Technology Research and Development Program of China (Grant No. 2006AA10A109-4) and the National Basic Research Program (Grant No. 2004CB117300)

About this article

Cite this article

Tu, L., Zhang, X., Liu, D. et al. Suitable internal control genes for qRT-PCR normalization in cotton fiber development and somatic embryogenesis. Chin. Sci. Bull. 52, 3110–3117 (2007). https://doi.org/10.1007/s11434-007-0461-0

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11434-007-0461-0

Keywords

  • cotton
  • fiber development
  • housekeeping genes
  • internal control
  • real-time PCR
  • somatic embryogenesis