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

Abstract

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 (EF1a, ACT, a-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, a-TUB and EF1a 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.

Isolation and characterization of three cold acclimation-responsive dehydrin genes from Eucalyptus globulus

“…Firststrand cDNA was synthesized by reverse transcription using the High-Capacity cDNA Archive Kit (Applied Biosystems, USA) according to the manufacturer's instructions. The PCR mixture, qRT-PCRs conditions, and two housekeeping genes (UBC and a-TUB) were used as described by Fernández et al (2010). Forward and reverse primer pairs used to amplify UBC, α-TUB, and EuglDHN genes, respectively, were: UBC-F/R: 5′-GACGGACAGGAACAAGTATGAGAC-3′ and 5′-CCCTCCACGGAATAATGATCGC-3′; α-TUB-F/R:…”

“…Two growth chamber assays were established. Temperature and photoperiod were changed according to the growth chamber assay reported by Fernández et al (2010). In the first growth chamber assay, four genotypes were studied.…”

Validation of reference genes from Eucalyptus spp. under different stress conditions

“…de Almeida et al [30] aimed to identify endogenous genes for gene expression studies associated with adventitious rooting in E. globulus microcuttings because low rooting efficiency is a problem for the cloning and multiplication of this species. Fernández et al [28] attempted to identify normaliser genes in E. globulus for gene expression studies related to cold acclimation. Similarly, Boava et al [29] sought to identify normaliser genes for studies of Urograndis hybrid resistance to leaf rust caused by Puccinia psidii .…”

Transcriptome Profile in Response to Frost Tolerance in Eucalyptus globulus

“…Growth chamber assays were established to assess the CA of both genotypes. Temperature and photoperiod were changed according to the growth chamber assay reported by Fernández et al (2010). Aislapool box containing the ramets were placed in the growth chamber and subjected to four treatments.…”

“…First-strand cDNA was synthesized by reverse transcription using High Capacity cDNA Archive kit (Applied Biosystems, USA) according to the manufacturer's instructions. The PCR mixture, qRT-PCR conditions, and two housekeeping genes (UBC and a-TUB) were used as described by Fernández et al (2010). Forward and reverse primer pairs used to amplify the candidate genes were designed using the Primer Express software: DHN1-F/ R: 5′-ATGGGCGGCGACGATGAG-3′ and 5′-CCCCTTCT TCTCCTGCTGCT-3′; DHN10-F/R: 5′-ACAGCAGCAGCA GCGACAG-3′ and 5′-TAGAGTGGACGAGGAGGACG AAC-3′; OEP-F/R: 5′-GCAAGAAAGCATTGGGAACAGT G-3′ and 5′-CCAGCAAGAATAACGGAGACAGG-3′; LTP-F/R: 5′-GGCAGCATCTCTGGCATCAAC-3′ and 5′-CCTT CCTTCACGATCTTCACTTCAC-3′; VDAC1-F/R: 5′-TGCTCCGCTCCGCCCTTC-3′ and 5′-TGGTCGCTCTGG TAGTCCTTGTAG-3′; TIL-F/R: 5′-CCTGGTGGCTCAAG TCCCTTATTG-3′ and 5′-CACATCACTCATCAACGCAG AACTC-3′; PCP-F/R: 5′-GCATTCTTCACTGCATCAGC-3′ and 5′-GCAAAGGATTCCATGCAAGA-3′; EIF-5A-F/R: 5′-CAGCAGGCCGGCACCATCC-3′ and 5′-TTCCAACA AAATGACACTTAGCATGGC-3′; ARP-F/R: 5′-ATGAGC ACCACGAGAAGAAGGAG-3′ and 5′ GCCAACACGGAT GTCAAGAAGAG-3′; CAX-F/R: 5′-GAGTCGGCGTCG GCTTCG-3′ and 5′-GGCGTGTTCTGTTGCGTTCC-3′.…”