Validating internal controls for quantitative plant gene expression
The goal of our studies was to detect changes associated with seasonal development and tree aging for several regulatory genes.We therefore undertook a study to compare the stability of several potential control genes.
This is an open-access article distributed under the terms of the Creative Commons Attribution License ( which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.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.RESULTS: Using 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.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.BACKGROUND: Real-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.
More rigorous methods will be needed as q RT-PCR is increasingly applied to study of regulatory genes, and for verifying patterns observed in microarray experiments.
In this study, we used real-time q RT-PCR to examine the expression of 10 housekeeping genes in a diversity of poplar (, cottonwood hybrid) tissues collected at different developmental stages, and at different times of the year.
Until recently, internal controls (often referred to as housekeeping or maintenance genes), were selected based on stability of expression in qualitative studies (e.g., visual examination of RNA gel-blots), via low-sensitivity assays such as densitometry of hybridized blots, or via semi-quantitative RT-PCR.
None of these will be adequate for identifying reliable internal controls for real-time q RT-PCR.
It is difficult to quantify small amounts of RNA, and variation in RT and PCR reaction efficiencies are not accounted for by this method.