Reference Genes for High-Throughput Quantitative Reverse Transcription–PCR Analysis of Gene Expression in Organs and Tissues of Eucalyptus Grown in Various Environmental Conditions

Abstract

Interest in the genomics of Eucalyptus has skyrocketed thanks to the recent sequencing of the genome of Eucalyptus grandis and to a growing number of large-scale transcriptomic studies. Quantitative reverse transcription-PCR (RT-PCR) is the method of choice for gene expression analysis and can now also be used as a high-throughput method. The selection of appropriate internal controls is becoming of utmost importance to ensure accurate expression results in Eucalyptus. To this end, we selected 21 candidate reference genes and used high-throughput microfluidic dynamic arrays to assess their expression among a large panel of developmental and environmental conditions with a special focus on wood-forming tissues. We analyzed the expression stability of these genes by using three distinct statistical algorithms (geNorm, NormFinder and ΔCt), and used principal component analysis to compare methods and rankings. We showed that the most stable genes identified depended not only on the panel of biological samples considered but also on the statistical method used. We then developed a comprehensive integration of the rankings generated by the three methods and identified the optimal reference genes for 17 distinct experimental sets covering 13 organs and tissues, as well as various developmental and environmental conditions. The expression patterns of Eucalyptus master genes EgMYB1 and EgMYB2 experimentally validated our selection. Our findings provide an important resource for the selection of appropriate reference genes for accurate and reliable normalization of gene expression data in the organs and tissues of Eucalyptus trees grown in a range of conditions including abiotic stresses.

Evaluation of Reference Genes for Accurate Normalization of Gene Expression for Real Time-Quantitative PCR in Pyrus pyrifolia Using Different Tissue Samples and Seasonal Conditions

“…Ct values of the newly added TIP _utr and SAND _utr were relatively higher by two to five cycles compared to ACT or GAP gene regions traditionally used. Similar results were also reported in Eucalyptus , papaya, grapevine, hop and banana [9], [21], [33], [34], [37]. For bTUB or HIS gene homologue, Ct values fluctuated among genes suggesting different levels of expression within homologous genes (Figure 1).…”

“…For the new three genes tested ( SAND , TIP and ANX ), amino acid identities were relatively low (about 70%). Similar identities were reported in Eucalyptus or tomato orthologous genes [9], [22].…”

“…Functional studies in yeast coupled with protein characterization in silico [32] suggested its roles in vacuoles or lysosomes. Although genes for SAND homologous proteins are isolated and their gene expression stabilities evaluated using plant cells or tissues [6], [9], [21], [22], [33], [34], no functional studies have been done. TIP41 ( T AP42- i nteracting p rotein of 41 kDa) is an accessory protein regulating the activity of protein phosphatase through interaction with a phosphatase inhibitor protein TAP42 in yeast [35].…”

“…Recently, the limitation of RT-qPCR in its ability to assess only a limited number of genes have been overcome with the development of the microfluidic technology that allows for high-throughput measurement of gene expression using dynamic arrays [8]. This microfluidic technology, which allows 9,216 simultaneous real-time PCR transcript quantification per single run has now been extended to studies in plants such as Eucalyptus [9]. Another way to accomplish high throughput RT-qPCR has been proposed by using nanoliter plate with 3,072 sample holes [10].…”

“…Several algorithms including geNorm [2], NormFinder [16], BestKeeper [17], ΔCt [18], qBasePlus [19], RefFinder [20], as well as single-factor analysis of variance (ANOVA) and linear regression analysis [14] have been used to evaluate the expression stability of reference genes. In recent times, studies on reference genes stability have been reported in plants including the model, crop and woody species [6], [9], [15], [21], [22], [23], [24], though these studies are relatively few compared to similar studies in animals or humans. The results of these studies suggested that expression stability of reference gene candidates varies depending on the plant source materials tested and a need for multiple reference genes for accurate evaluation [2], [15].…”