Abstract
BackgroundGene 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.ResultsA 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.ConclusionsThe 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.
Highlights
Gene expression analysis using quantitative reverse transcription 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
Assessment of RNA integrity and verification of PCR products Agarose gel electrophoresis images of total RNA extracted from all olive samples revealed two distinct bands representing 18S- and 28S- RNA bands and the RNA integrity number (RIN) values for all samples ranged from 8.3-9.9
Amplification of the eight candidate reference genes by PCR revealed products of the expected sizes (Additional file 1) and subsequent sequencing of these products revealed that all amplified fragments were identical to sequences used for designing primers for the reference genes (Table 1)
Summary
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. Reference genes have recently been suggested for olive tissues, combined/independent analysis on different cultivars has not yet been tested. An assessment of reference genes was required to validate the recent findings and select stably expressed genes across different olive cultivars. Recent reports have shown that the most commonly used traditional reference genes may be inappropriate for normalization in qPCR experiments due to their expression variability under different experimental conditions [8,13]. For accurate analysis of RNA transcription it is crucial to choose reference genes that have been shown to be minimally regulated in a given species, in a given organs/tissues, at a given developmental period and under specific environmental conditions
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