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


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.

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This research was supported by Genomica Forestal S.A. and a scholarship from Conicyt to MF. Gratitude is expressed to Valeria Neira for technical assistance. We thank Dr. Leon Bravo for moving and establishing the growth chamber assay.

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Correspondence to Sofia Valenzuela.

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Communicated by F. M. Cánovas.

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Fernández, M., Villarroel, C., Balbontín, C. et al. Validation of reference genes for real-time qRT-PCR normalization during cold acclimation in Eucalyptus globulus . Trees 24, 1109–1116 (2010).

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