Abstract
BackgroundReal-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed.ResultsUsing real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied.ConclusionOur results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments.
Highlights
Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies
Real-time PCR technology has removed many of the difficulties associated with quantitative gene expression studies [1], and realtime quantitative RT-PCR is rapidly being adopted as a standard method for in-depth expression studies, including studies of alternative splicing, verification of microarray expression results, and molecular diagnostics [2-5]
Searches of the literature revealed that members of all classes have been used as internal controls for studies of plant gene expression using RNA gel blots or RT-PCR assays
Summary
Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. Selection of an appropriate normalization method is crucial for reliable quantitative gene expression results [1,6]. The purpose of normalization is to correct for non-specific variation, such as differences in RNA quantity and quality, which can affect efficiencies of the RT and PCR reactions. It is difficult to quantify small amounts of RNA, and variation in RT and PCR reaction efficiencies are not accounted for by this method. The most commonly used method is relative quantitation, whereby gene expression level is normalized to that of an internal reference gene. While this avoids the problems and limitations of absolute quantitation, selection of a proper internal control–gene expressed at a nearly constant level in all tissue samples being investigated–is required. The consequences may be that only gross changes in expression level are declared statistically significant, or that patterns of expression are erroneously characterized
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