Selection of reference genes for real-time quantitative PCR studies of kumquat in various tissues and under abiotic stress

The proper selection of stable reference genes for real-time quantitative PCR (RT-qPCR) can help to reliably assess gene expression results under specific experimental conditions. The present study evaluated the stability of 16 candidate reference genes of kumquat using the methods of geNorm, Normfinder, and Bestkeeper in different tissues of kumquat (Fortunella crassifolia Swingle) under some type of abiotic stress. The results showed that homologous genes should be taken into account for normalization in RT-qPCR studies. For the three specific types of stresses, i.e., salt stress, drought stress, and heavy metal stress, ACT6, ACT8, and ACT7 should be selected as internal controls, respectively. For low-temperature stress, the use of multiple reference genes for normalization may be optimal, such as ACT7+ACT9 (according to geNorm) or ACT7+TUB4 (according to Normfinder). We suggest that ACT7 alone or the combination of ACT7+ACT1+EF1-alpha or ACT1+ACT8 can meet the requirements for the normalization of RT-qPCR studies under the experimental conditions tested here. Although some commonly used reference genes (TUA1, TUB1, TUB4, CYP, GAPDH, etc.) showed much higher variability, some are still useful in combinations under specific conditions. (C) 2014 Published by Elsevier B.V.