Selection and validation of reference genes for quantitative Real-Time PCR in Arabis alpina

RESEARCH ARTICLE Selection and validation of reference genes for quantitative Real-Time PCR in Arabis alpina Lisa Stephan1, Vicky Tilmes2, Martin Hülskamp ID1* 1 Botanical Institute, Biocenter, Cologne University, Cologne, Germany, 2 Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany * a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Stephan L, Tilmes V, Hülskamp M (2019) Selection and validation of reference genes for quantitative Real-Time PCR in Arabis alpina. PLoS ONE 14(3): e0211172. https://doi.org/10.1371/ journal.pone.0211172 Editor: Miguel A. Blázquez, Instituto de Biologia Molecular y Celular de Plantas, SPAIN Received: January 8, 2019 Accepted: February 19, 2019 Abstract Arabis alpina is a perennial arctic-alpine plant and an upcoming model organism for genetics and molecular biology for the Brassicaceae family. One essential method for most molecular approaches is the analysis of gene expression by reverse-transcription quantitative RealTime PCR (RT-qPCR). For the normalisation of expression data in RT-qPCR experiments, it is essential to use reliable reference genes that are not affected under a wide range of conditions. In this study we establish a set of 15 A. alpina reference genes that were tested under different conditions including cold, drought, heat, salt and gibberellic acid treatments. Data analyses with geNORM, BestKeeper and NormFinder revealed the most stable reference genes for the tested conditions: RAN3, HCF and PSB33 are most suitable for cold treatments; UBQ10 and TUA5 for drought; RAN3, PSB33 and EIF4a for heat; CAC, TUA5, ACTIN 2 and PSB33 for salt and PSB33 and TUA5 for gibberellic acid treatments. CAC and ACTIN 2 showed the least variation over all tested samples. In addition, we show that two reference genes are sufficient to normalize RT-qPCR data under our treatment conditions. In future studies, these reference genes can be used for an adequate normalisation and thus help to generate high quality RT-qPCR data in A. alpina. Published: March 4, 2019 Copyright: © 2019 Stephan et al. 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 author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Funding: This work was funded by the Deutsche Forschungsgemeinschaft (https://www.dfg.de/) to MH and by the International Max Planck Research School to LS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Introduction Recently, A. alpina has been established as a new model system in the Brassicaceae family [1,2]. It is native to mountains and arctic-alpine habitats [3,4] and combines several features enabling genetic and molecular studies: it is diploid, self-fertile, has a small and sequenced genome and can be transformed with Agrobacterium tumefaciens [1]. A. alpina has an evolutionary distance to A. thaliana of about 26 to 40 million years [4,5]. This facilitates functional comparisons of biological processes, as orthologous genes can be identified by sequence similarity and synteny [6]. Most molecular studies require quantitative analyses of the expression of genes of interest by reverse-transcription quantitative Real-Time PCR (RT-qPCR). For proper comparisons of expression levels, the expression data of the genes under study are normalized using genes as a reference that show no or very little variation under different conditions. In 2009, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) PLOS ONE | https://doi.org/10.1371/journal.pone.0211172 March 4, 2019 1 / 13 Reference RT-qPCR primers for Arabis alpina guidelines were published, with the aim to provide a consensus on correct performance and interpretation of RT-qPCR experiments [7]. These guidelines should ensure that the normalisation enables the comparison of transcripts in different samples by correcting variations in yields of extraction and reverse transcription and the efficiency of amplification. A pre-requisite for any RT-qPCR analysis are suitable primer sets for reference genes that are thoroughly tested. These need to fulfil various requirements: primers should create a specific amplicon of 80 to 200 bp, without creating primer dimers. The amplification should be carried out with close to 100% efficiency and show a linear standard curve with a correlation of more than 0.99. In general, there should be minimal variation between replicates, indicating consistent performance of the primers. In this study we established primer pairs for 15 reference genes that can be used for future RT-qPCR studies in A. alpina: ADENOSINE TRIPHOSPHATASE (ATPase), THIOREDOXIN, HIGH CHLOROPHYLL FLUORESCENCE 164 (HCF), EUKARYOTIC TRANSLATION INITIATION FACTOR 4A1 (EIF4a), RAN GTPASE 3 (RAN3), UBIQUITIN 10 (UBQ10), ACTIN 2, PHOTOSYSTEM B PROTEIN 33 (PSB33), HISTONE H3, NAD(P)H PLASTOQUINONE DEHYDROGENASE COMPLEX SUBUNIT O (NdhO), TUBULIN ALPHA 5 (TUA5), 18s RIBOSOMAL RNA (18srRNA), CLATHRIN ADAPTOR COMPLEX MEDIUM SUBUNIT (CAC), SAND family protein (SAND) and HEAT SHOCK PROTEIN 81.2/90 (HSP81.2/90). The primers were thoroughly tested, and reference genes were evaluated for variations in their expression under different conditions including cold, drought, heat and salt in whole seedlings and gibberellic acid (GA) treatments in leaves. Using genes specifically responding to the different treatment, we demonstrate the impact of normalization with our reference genes. Results Selection and validation of reference genes To compile a set of suitable reference genes, we pursued three approaches: first, we selected A. thaliana genes that are known to show little variation under different conditions. Corresponding orthologs in A. alpina were then identified by sequence similarity and synteny. Second, we identified genes which show stable expression in A. alpina over an extended period of time. Third, we included a well-established reference gene from A. alpina from former studies. Thus, we created a set of 15 reference genes (Table 1), including nine orthologs to A. thaliana reference genes, five novel reference genes and RAN3, a known reference gene for RT-qPCR in A. alpina [1]. We first amplified the gene fragments and verified the amplicon by sequencing. Additionally, we analysed the melting curves to exclude unspecific products and/or primer dimers (S1 Fig). Subsequently, we determined the primer efficiencies and correlation coefficients to demonstrate the quality of the primer pairs (Table 2, S2 Fig). All reference gene primers displayed an efficiency between 96.42 and 10 (...truncated)