, Volume 24, Issue 6, pp 1109–1116 | Cite as

Validation of reference genes for real-time qRT-PCR normalization during cold acclimation in Eucalyptus globulus

  • Marta Fernández
  • Carlos Villarroel
  • Cristián Balbontín
  • Sofia Valenzuela
Original Paper


During the last few years, many studies have directed their efforts at elucidating the molecular mechanisms that regulate plant response to cold stress using gene expression analysis. Quantitative real-time qRT-PCR has great advantages compared to traditional transcriptional detection methods due to its high sensibility, reproducibility, and specificity for the detection of low quantities of RNA. However, this technique requires the use of one or several housekeeping genes. In this work, the expression stabilities of six housekeeping genes (EF1α, ACT, α-TUB, PDF, SAND, and UBC) during the cold acclimation of E. globulus plants was analyzed. An ELIP gene that responds to photooxidative stress caused by light and cold stress was used as the target gene to identify the most suitable internal control for normalizing real-time qRT-PCR. Two additional genes involved in the ABA biosynthesis pathway (NCED) and sugar metabolism (GS) were analyzed with the most stable internal control genes in order to check the results found with the ELIP gene. The expressions of UBC, α-TUB and EF1α were the most stable across acclimation and de-acclimation treatments. The expressions of the other housekeeping genes tested varied depending upon the conditions. The relative quantification of ELIP changed according to identities and the number of reference genes used, thus demonstrating the importance of selecting an appropriate number of reference genes in order to achieve an accurate and reliable normalization of gene expression during cold acclimation in E. globulus.


Eucalyptus globulus Cold acclimation Reference genes Real-time qRT-PCR Normalization ELIPs 

Supplementary material

468_2010_483_MOESM1_ESM.doc (38 kb)
Supplementary material 1 (DOC 37 kb)


  1. Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2:169–193Google Scholar
  2. Chang S, Puryear J, Cairney J (1993) A simple method for isolating RNA from pine trees. Plant Mol Biol Rep 11:113–116CrossRefGoogle Scholar
  3. Costa e Silva F, Shvaleva A, Broetto F, Ortuño MF, Rodrigues ML, Almeida MH, Chaves MM, Pereira JS (2008) Acclimation to short-term low temperatures in two Eucalyptus globulus clones with contrasting drought resistance. Tree Physiol 29:77–86CrossRefPubMedGoogle Scholar
  4. 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 139:5–17CrossRefPubMedGoogle Scholar
  5. Dhanaraj AL, Slovin JP, Rowland LJ (2004) Analysis of gene expression associated with cold acclimation in blueberry floral buds using expressed sequence tags. Plant Sci 166:863–872CrossRefGoogle Scholar
  6. Dhanaraj AL, Alkharouf NW, Beard HS, Chouikha IB, Matthews BF, Wei H, Arora R, Rowland LJ (2007) Major differences observed in transcript profiles of blueberry during cold acclimation under field and cold room conditions. Planta 225:735–751CrossRefPubMedGoogle Scholar
  7. Fernández M, Valenzuela S, Balocchi C (2006) RAPD and freezing resistance in Eucalyptus globulus. Electron J Biotechnol 9:303–309Google Scholar
  8. Houde M, Belcaid M, Ouellet F, Danyluk J, Monroy AF, Dryanova A, Gulick P, Bergeron A, Laroche A, Links MG, MacCarthy L, Crosby WL, Sarhan F (2006) Wheat EST resources for functional genomics of abiotic stress. BMC Genomics 7:149CrossRefPubMedGoogle Scholar
  9. Jain M, Nijhawan A, Tyagi AK, Khurana JP (2006) Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun 345:646–665Google Scholar
  10. Jeong YM, Mun JH, Lee I, Woo JC, Hong CB, Kim SG (2006) Distinct roles of the first introns on the expression of Arabidopsis profiling gene family members. Plant Physiol 140:196–209CrossRefPubMedGoogle Scholar
  11. Joosen RVL, Lammers M, Balk PA, Brønnum P, Konings MC, Perks M, Stattin E, Van Wordragen MF, Van der Geest AL (2006) Correlating gene expression programs to physiological parameters and environmental conditions during cold acclimation of pine (Pinus sylvestris). Tree Physiol 26:1297–1313PubMedGoogle Scholar
  12. Keller G, Marchal T, SanClemente H, Navarro M, Ladouce N, Wincker P, Couloux A, Teulières C, Marque C (2009) Development and functional annotation of an 11,303-EST collection from Eucalyptus for studies of cold stress. Tree Genet Genomes 5:317–327Google Scholar
  13. Li L, Xu J, Xu ZH, Xue HW (2005) Brassinosteroids stimulate plant tropisms through modulation of polar auxin transport in Brassica and Arabidopsis. Plant Cell 17:2738–2753CrossRefPubMedGoogle Scholar
  14. Libault M, Thibivilliers S, Radwan O, Clough SJ, Stacey G (2008) Identification of four soybean reference genes for gene expression normalization. Plant Genome 1:44–54CrossRefGoogle Scholar
  15. Miranda I, Pereira H (2002) Variation of pulpwood quality with provenances and site in Eucalyptus globulus. Ann For Sci 59:283–291CrossRefGoogle Scholar
  16. Moraga P, Escobar R, Valenzuela S (2006) Resistance to freezing in three Eucalyptus globulus Labill subspecies. Electron J Biotechnol 9:310–314Google Scholar
  17. Nicot M, 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(421):2907–2914Google Scholar
  18. Peng Y, Lin W, Wei H, Krebs SL, Arora R (2008) Phylogenetic analysis and seasonal cold acclimation-associated expression of early light-induced protein genes of Rhododendron catawbiense. Physiol Plantarum 132:44–52Google Scholar
  19. Rasmussen-Poblete S, Valdés J, Gamboa M, Valenzuela P, Krauskopf E (2008) Generation and analysis of an Eucalyptus globulus cDNA library constructed from seedlings subjected to low temperature conditions. Electronic J Biotech 11:2Google Scholar
  20. Sekalska B, Ciechanowicz A, Dolegowska B, Naruszewicz M (2006) Optimized RT-PCR method for assaying expression of monocyte chemotactic protein type 1 (MCP-1) in rabbit aorta. Biochem Genet 44:133–143CrossRefPubMedGoogle Scholar
  21. Soitamo AJ, Piippo M, Allahverdiyeva Y, Battchikova N, Aro E (2008) Light has a specific role in modulating Arabidopsis gene expression at low temperature. BMC Plant Biol 8:13Google Scholar
  22. Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, Grisar T, Igout A, Heinen E (1999) Housekeeping genes as internal standards: use and limits. J Biotechnol 75:291–295CrossRefPubMedGoogle Scholar
  23. Travert S, Valeria L, Fourasté I, Boudet AM, Teuliéres C (1997) Enrichment in specific soluble sugars of two Eucalyptus cell suspension cultures by various treatments enhances their frost tolerance via a non colligative mechanism. Plant Physiol 114:1433–1442PubMedGoogle Scholar
  24. 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:RESEARCH0034Google Scholar
  25. Zarter CR, Adams WW III, Ebbert V, Adamska I, Jansson S, Demmig-Adams B (2006) Winter acclimation of PsbS and related proteins in the evergreen Arctostaphylos uva-ursi as influenced by altitude and light environment. Plant Cell Environ 29:869–878CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Marta Fernández
    • 1
    • 2
  • Carlos Villarroel
    • 2
  • Cristián Balbontín
    • 3
  • Sofia Valenzuela
    • 1
    • 2
    • 3
  1. 1.Faculty of Forest SciencesUniversity of ConcepciónConcepciónChile
  2. 2.Biotechnology CenterUniversity of ConcepciónConcepciónChile
  3. 3.Genomica Forestal S.A.ConcepciónChile

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