Abstract
The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR) method for gene expression analysis requires one or several reference gene(s) acting as normalization factor(s). In order to facilitate gene expression studies in sugarcane (Saccharum officinarum), a non-model plant with limited genome information, the stability of 13 candidate reference genes was evaluated. The geNorm, NormFinder and deltaCt methods were used for selecting stably expressed internal controls across different tissues and under various experimental treatments. These results revealed that, among these 13 candidate reference genes, GAPDH, eEF-1a and eIF-4α were the most stable and suitable for use as normalization factors across all various experimental samples. In addition, APRT could be a candidate for examining the relationship between gene copy number and transcript levels in sugarcane tissue samples. According to the results evaluated by geNorm, combining CUL and eEF-1α in hormone treatment experiments; CAC and CUL in abiotic stress tests; GAPDH, eEF-1a and CUL in all treatment samples plus CAC, CUL, APRT and TIPS-41 in cultivar tissues as groups for normalization would lead to more accurate and reliable expression quantification in sugarcane. This is the first systematic validation of reference genes for quantification of transcript expression profiles in sugarcane. This study should provide useful information for selecting reference genes for more accurate quantification of gene expression in sugarcane and other plant species.
Highlights
Real time quantitative reverse transcription-PCR is increasingly used in gene expression analysis owing to its simple, reproducible and high-throughput features. qRT-PCR provides a useful and rapid means of understanding gene expression in living organisms by measuring the expression of target genes across different samples
In order to obtain a reliable analysis of gene expression by qRT-PCR, one or several reference genes should serve as the internal control to normalize and monitor the expression variation between samples and reactions [2,3,4]
A total of 13 candidate reference genes were selected in sugarcane or in other plant species for evaluation on the basis of their stable expression across developmental stages and/or abiotic stresses
Summary
Real time quantitative reverse transcription-PCR (qRT-PCR) is increasingly used in gene expression analysis owing to its simple, reproducible and high-throughput features. qRT-PCR provides a useful and rapid means of understanding gene expression in living organisms by measuring the expression of target genes across different samples. In order to obtain a reliable analysis of gene expression by qRT-PCR, one or several reference genes should serve as the internal control to normalize and monitor the expression variation between samples and reactions [2,3,4]. The expression of these reference genes should remain stable under various experimental treatments and/ or at different stages of development and growth periods [2,3,4]. A suitable reference gene for performing qRT-PCR analysis should: (i) have stable expression across all or most of the samples analyzed; (ii) have no association with any pseudogene, to avoid the amplification of non-functional gene family members;
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