title: 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.
authors.affiliation: Embrapa Clima Temperado, Rodovia BR 396, Km 78, Caixa Postal 403, Pelotas, RS, CEP 96001-970, Brazil.
authors.forename: Joyce Moura
authors.initials: JM
authors.lastname: Borowski
authors.forename: Vanessa
authors.initials: V
authors.lastname: Galli
authors.forename: Rafael da Silva
authors.initials: Rda S
authors.lastname: Messias
authors.forename: Ellen Cristina
authors.initials: EC
authors.lastname: Perin
authors.forename: Julieti Hugh
authors.initials: JH
authors.lastname: Buss
authors.forename: Sérgio Delmar
authors.initials: SD
authors.lastname: dos Anjos e Silva
authors.forename: Cesar Valmor
authors.initials: CV
authors.lastname: Rombaldi
mesh_terms: D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D014644:Genetic Variation; D018545:Lactuca / Q000235:genetics* / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013312:Stress, Physiological / Q000235:genetics*; D059467:Transcriptome
chemical_list: D035683:MicroRNAs; D010940:Plant Proteins; D018749:RNA, Plant