Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR

Abstract

Background: Gene expression analysis using quantitative reverse transcription PCR (qRT-PCR) is a robust method wherein the expression levels of target genes are normalised using internal control genes, known as reference genes, to derive changes in gene expression levels. Although reference genes have recently been suggested for olive tissues, combined/independent analysis on different cultivars has not yet been tested. Therefore, an assessment of reference genes was required to validate the recent findings and select stably expressed genes across different olive cultivars.

Results: A total of eight candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine/threonine-protein phosphatase catalytic subunit (PP2A), elongation factor 1 alpha (EF1-alpha), polyubiquitin (OUB2), aquaporin tonoplast intrinsic protein (TIP2), tubulin alpha (TUBA), 60S ribosomal protein L18-3 (60S RBP L18-3) and polypyrimidine tract-binding protein homolog 3 (PTB)] were chosen based on their stability in olive tissues as well as in other plants. Expression stability was examined by qRT-PCR across 12 biological samples, representing mesocarp tissues at various developmental stages in three different olive cultivars, Barnea, Frantoio and Picual, independently and together during the 2009 season with two software programs, GeNorm and BestKeeper. Both software packages identified GAPDH, EF1-alpha and PP2A as the three most stable reference genes across the three cultivars and in the cultivar, Barnea. GAPDH, EF1-alpha and 60S RBP L18-3 were found to be most stable reference genes in the cultivar Frantoio while 60S RBP L18-3, OUB2 and PP2A were found to be most stable reference genes in the cultivar Picual.

Conclusions: The analyses of expression stability of reference genes using qRT-PCR revealed that GAPDH, EF1-alpha, PP2A, 60S RBP L18-3 and OUB2 are suitable reference genes for expression analysis in developing Olea europaea mesocarp tissues, displaying the highest level of expression stability across three different olive cultivars, Barnea, Frantoio and Picual, however the combination of the three most stable reference genes do vary amongst individual cultivars. This study will provide guidance to other researchers to select reference genes for normalization against target genes by qPCR across tissues obtained from the mesocarp region of the olive fruit in the cultivars, Barnea, Frantoio and Picual.

Figure 1

Expression levels of eight candidate reference genes. The values are given as real-time PCR quantification cycle (Cq) values for individual reference genes in a total of 12 samples (in duplicate) from the individual timepoints in the 2009 (96, 109, 116 and 136 DAF) crop season from cultivars Barnea, Frantoio and Picual. The boxes represent the 25^th and 75^th percentiles and the line within the boxes represents the median. The whiskers indicate the range of Cq values of the data of the 24 samples per reference gene.

Figure 2

Validation of candidate reference genes in Barnea, Frantoio and Picual as a whole using GeNorm algorithm in qBase Plus. (A) Average expression stability values (M) of the eight reference genes plotted from least stable (left) to most stable (right). The M value was calculated for each gene and the least stable gene with the highest M value was excluded from the next calculation round. (B) Pairwise variation analysis (V_n/V_n+1) between the normalisation factors NFn and NFn + 1 to determine the optimal number of reference genes to be used for normalisation against target genes. GeNorm V calculates the normalisation factor (NF_n) by calculating the geometric mean of the expression levels of the stable most reference genes by step-wise inclusion of a less stable gene.

Figure 3

Validation of candidate reference genes in Barnea, Frantoio and Picual independently using GeNorm algorithm in qBase Plus. (A) Average expression stability values (M) of the eight reference genes plotted from least stable (left) to most stable (right) in Barnea (A), Frantoio (C) and Picual (E) (B) Pairwise variation analysis (V_n/V_n+1) between the normalisation factors NFn and NFn + 1 to determine the optimal number of reference genes to be used for normalisation against target genesin Barnea (B), Frantoio (D) and Picual (F).