Comparison of predictive methods and biological validation for qPCR reference genes in sunflower leaf senescence transcript analysis

Abstract

The selection and validation of reference genes constitute a key point for gene expression analysis based on qPCR, requiring efficient normalization approaches. In this work, the expression profiles of eight genes were evaluated to identify novel reference genes for transcriptional studies associated to the senescence process in sunflower. Three alternative strategies were applied for the evaluation of gene expression stability in leaves of different ages and exposed to different treatments affecting the senescence process: algorithms implemented in geNorm, BestKeeper software, and the fitting of a statistical linear mixed model (LMModel). The results show that geNorm suggested the use of all combined genes, although identifying α-TUB1 as the most stable expressing gene. BestKeeper revealed α-TUB and β-TUB as stable genes, scoring β-TUB as the most stable one. The statistical LMModel identified α-TUB, actin, PEP, and EF-1α as stable genes in this order. The model-based approximation allows not only the estimation of systematic changes in gene expression, but also the identification of sources of random variation through the estimation of variance components, considering the experimental design applied. Validation of α-TUB and EF-1α as reference genes for expression studies of three sunflower senescence associated genes showed that the first one was more stable for the assayed conditions. We conclude that, when biological replicates are available, LMModel allows a more reliable selection under the assayed conditions. This study represents the first analysis of identification and validation of genuine reference genes for use as internal control in qPCR expression studies in sunflower, experimentally validated throughout six different controlled leaf senescence conditions.

Functional Approaches to Study Leaf Senescence in Sunflower

“…Average Cq of analyzed SAGs genes normalizing against a-TUB as RG. Error bars show standard deviation (Fernandez et al 2011).…”

“…Specific oligonucleotides were designed to amplify fragments of approximately 150 bp for further evaluation by quantitative PCR. In a previous study, we have reported the evaluation and identification of a panel of eight reference genes for their application to transcriptional analysis of the leaf senescence process, thus enabling the use of genuine reference genes in ongoing expression studies (Fernandez et al 2011). Exploratory studies of senescence by qPCR comparing two treatments which affect the rate of leaf senescence were performed: water stress and head excision, relative to a control condition.…”

“…Table 1. Average Cq and CV value for R2, D3 and D4 genes and the two best ranked RGs for three biological replicates per treatment (Fernandez et al 2011). …”

“…Two of the selected genes, a gamma vacuolar processing enzyme (AN At5g60360) (D3 gene) involved in the maturation and activation of vacuolar proteins and an aleurain protease AALP, (AN At1g18210) (D4 gene), belonging to the cystein-protease family are classified in the group of macromolecular degradation and recycling; the third gene, a calcium binding protein (AN At4g32940) (R2 gene) belongs to the group of regulatory genes (Gepstein et al 2003). Furthermore two reference genes were evaluated against these conditions for relative expression studies, Elongation Factor 1-α (AN) and α-Tubuline, selected from a previous study of the performance of different reference genes against these experimental conditions in sunflower (Fernandez et al 2011). Alfa tubuline (α-Tubuline) showed the most stable behavior; therefore, it was selected as internal control in further analysis of expression of these SAGs (Figure 1).…”

“…Although, molecular studies focused on the onset of the senescence process in sunflower leaf are scarce (Fernandez et al 2003;Dezar et al 2005;Manavella et al 2006;Jobit et al 2007;Paniego et al 2007;Fernandez et al 2008;Manavella et al 2008;Peluffo et al 2010;Fernandez et al 2011). Thus, two different approaches are envisage for studying molecular events occurring during leaf senescence: the first strategy relays on the identification of sunflower SAGs based on a candidate gene approach while the second approach involves concerted gene expression studies based on high density oligonucleotide microarrays, whole transcriptome shotgun sequencing and microRNA detection by RNA-seq (Buermans et al 2010;Dhahbi et al 2011).…”