Reference Gene Selection for Real-Time Quantitative Polymerase Chain Reaction of mRNA Transcript Levels in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Abstract

The Chinese cabbage is a crop belonging to the family Cruciferae; very few studies have focused on the analysis of gene expression in this economically important crop. In this study, we used real-time quantitative polymerase chain reaction to identify the control genes that are the most stably expressed in a given set of tissues and under conditions of drought stress and downy mildew infection. We characterized the transcript stability of nine candidate reference genes, namely, those encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (Ubc), elongation-factor-1-α (EF-1-α), adenine phosphoribosyltransferase (Apr), clathrin, cyclophilin (Cyp), tubulin (Tub), Actin, and 18s rRNA, using the geNorm and Normfinder software programs. The results of a geNorm analysis indicated that EF-1-α and Apr were the most suitable reference genes among the given set of tissues and that GAPDH and Ubc were the most stable genes under conditions of drought stress and downy mildew infection. Furthermore, the results of an analysis performed using the Normfinder software program indicated EF-1-α to be the best normalization factor in the given set of tissues and under conditions of downy mildew infection and GAPDH to be the best factor under drought stress conditions. The results of the geNorm analysis also helped determine the minimum number of genes required to calculate a reliable normalization factor. A study on the variability of expression of PSY expression showed that the relative quantification of this gene varied depending on the internal control and the number of internal controls used, thus highlighting the importance of the choice of internal controls in such experiments.