Selection of candidate reference genes for real-time PCR studies in lettuce under abiotic stresses

Abstract

The process of selection and validation of reference genes is the first step in studies of gene expression by real-time quantitative polymerase chain reaction (RT-qPCR). The genome of lettuce, the most popular leaf vegetable cultivated worldwide, has recently been sequenced; therefore, suitable reference genes for reliable results in RT-qPCR analyses are required. In the present study, 17 candidate reference genes were selected, and their expression stability in lettuce leaves under drought, salt, heavy metal, and UV-C irradiation conditions and under the application of abscisic acid (ABA) was evaluated using geNorm and NormFinder software. The candidate reference genes included protein-coding traditional and novel reference genes and microRNAs (miRNAs). The results indicate that the expression stability is dependent on the experimental conditions. The novel protein-coding reference genes were more suitable than the traditional reference genes under drought, UV-C irradiation, and heavy metal conditions and under the application of ABA. Only under salinity conditions were the traditional protein-coding reference genes more stable than the novel genes. In addition, the miRNAs, mainly MIR169, MIR171/170 and MIR172, were stably expressed under the abiotic stresses evaluated, representing a suitable alternative approach for gene expression data normalization. The expression of phenylalanine ammonia lyase (PAL) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) was used to further confirm the validated protein-coding reference genes, and the expression of MIR172 and MIR398 was used to confirm the validated miRNA genes, showing that the use of an inappropriate reference gene induces erroneous results. This work is the first survey of the stability of reference genes in lettuce and provides guidelines to obtain more accurate RT-qPCR results in lettuce studies.

Evaluation of reference genes for RT-qPCR studies in the leaves of rice seedlings under salt stress

“…For example, when evaluating the expression of PAL and HPPD genes in lettuce plants (Lactuca sativa L.), Borowski et al (2014) observed significant differences in the expression of these genes depending on both the reference gene and stress condition used. The results of this study confirm the importance of a specific choice of reference genes for transcript analyses by RT-qPCR.…”

Identification of reference genes for RT-qPCR analysis in peach genotypes with contrasting chilling requirements

“…RT-qPCR has become the most popular method for the quantification of transcript accumulation and must be preceded by validated normalization to prevent variations arising from non-biological events (Borowski et al, 2014). Questionable gene expression results occur when target genes are normalized against single reference genes without adequate justification and when non-validated reference genes are used (Bustin et al, 2009;Derveaux et al, 2010).…”

“…The purpose of these reference genes is to normalize the variations in RNA quantity and quality (Ponton et al, 2011). This validation requires the most suitable reference gene to be chosen to normalize the results for each species and under each experimental condition (Borowski et al, 2014).…”

“…A single reference gene is not universal for all experiments (Nicot et al, 2005;Borowski et al, 2014;Galli et al, 2015).…”

“…Most of these studies have been done with plants, such as wheat (Paolacci et al, 2009), barley (Burton et al, 2004), rice (Kim et al, 2003;Ding et al, 2004;Jain et al, 2006), potato (Nicot et al, 2005), soybean (Jian et al, 2008), grape (Reid et al, 2006), poplar (Brunner et al, 2004), tomato (Coker and Davies, 2003;Expósito-Rodríguez et al, 2008), coffee (BarsalobresCavallari et al, 2009), Arabidopsis thaliana (Czechowski et al, 2005;Remans et al, 2008), sugarcane (Iskandar et al, 2004), Eremosparton songoricum (Li et al, 2012), Lolium perenne (Lee et al, 2010), lettuce (Borowski et al, 2014), strawberry (Galli et al, 2015), and Salicornia europaea (Xiao et al, 2015).…”