Dataset Information

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

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 Real-Time 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.

SUBMITTER: Stephan L

PROVIDER: S-EPMC6398851 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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

Stephan Lisa L Tilmes Vicky V Hülskamp Martin M

PloS one 20190304 3

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 Real-Time 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 ...[more]

PMID: 30830921

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Project description:Lycoris aurea (L' Hér.) Herb, a perennial grass species, produces a unique variety of pharmacologically active Amaryllidaceae alkaloids. However, the key enzymes and their expression pattern involved in the biosynthesis of Amaryllidaceae alkaloids (especially for galanthamine) are far from being fully understood. Quantitative real-time polymerase chain reaction (qRT-PCR), a commonly used method for quantifying gene expression, requires stable reference genes to normalize its data. In this study, to choose the appropriate reference genes under different experimental conditions, 14 genes including YLS8 (mitosis protein YLS8), CYP2 (Cyclophilin 2), CYP 1 (Cyclophilin 1), TIP41 (TIP41-like protein), EXP2 (Expressed protein 2), PTBP1 (Polypyrimidine tract-binding protein 1), EXP1 (Expressed protein 1), PP2A (Serine/threonine-protein phosphatase 2A), ?-TUB (?-tubulin), ?-TUB (?-tubulin), EF1-? (Elongation factor 1-?), UBC (Ubiquitin-conjugating enzyme), ACT (Actin) and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were selected from the transcriptome datasets of L. aurea. And then, expressions of these genes were assessed by qRT-PCR in various tissues and the roots under different treatments. The expression stability of the 14 candidates was analyzed by three commonly used software programs (geNorm, NormFinder, and BestKeeper), and their results were further integrated into a comprehensive ranking based on the geometric mean. The results show the relatively stable genes for each subset as follows: (1) EXP1 and TIP41 for all samples; (2) UBC and EXP1 for NaCl stress; (3) PTBP1 and EXP1 for heat stress, polyethylene glycol (PEG) stress and ABA treatment; (4) UBC and CYP2 for cold stress; (5) PTBP1 and PP2A for sodium nitroprusside (SNP) treatment; (6) CYP1 and TIP41 for methyl jasmonate (MeJA) treatment; and (7) EXP1 and TIP41 for various tissues. The reliability of these results was further enhanced through comparison between part qRT-PCR result and RNA sequencing (RNA-seq) data. In summary, our results identified appropriate reference genes for qRT-PCR in L. aurea, and will facilitate gene expression studies under these conditions.

Project description:BACKGROUND:The use of stably expressed genes as normalizers has crucial role in accurate and reliable expression analysis estimated by quantitative real-time polymerase chain reaction (qPCR). Recent studies have shown that, the expression levels of common housekeeping genes are varying in different tissues and experimental conditions. The genomic DNA contamination in RNA samples is another important factor that also influence the interpretation of the data obtained from qPCR. It is estimated that the gDNA contamination in gene expression analysis lead to an overestimation of the RNA transcript level. The aim of this study was to validate the most stably expressed reference genes in two different tissues of Aeluropus littoralis-halophyte grass at salt stress and recovery condition. Also, a qPCR-based approach for monitoring contamination with gDNA was conducted. RESULTS:Ten candidate reference genes participating in different biological processes were analyzed in four groups of samples including root and leaf tissues, salt stress and recovery condition. To determine the most stably expressed reference genes, three statistical methods (geNorm, NormFinder and BestKeeper) were applied. According to results obtained, ten candidate reference genes were ranked based on the stability of their expression. Here, our results show that a set of four housekeeping genes (HKGs) e.g. RPS3, EF1A, GTF and RPS12 could be used as general reference genes for the all selected conditions and tissues. Also, four set of reference genes were proposed for each tissue and condition including: RPS3, EF1A and UBQ for salt stress and root samples; RPS3, EF1A, UBQ as well as GAPDH for recovery condition; U2SURP and GTF for leaf samples. Additionally, for assessing DNA contamination in RNA samples, a set of unique primers were designed based on the conserved region of ribosomal DNA (rDNA). The universality, specificity and sensitivity of these primer pairs were also evaluated in Poaceae. CONCLUSIONS:Overall, the sets of reference genes proposed in this study are ideal normalizers for qPCR analysis in A. littoralis transcriptome. The novel reference gene e.g. RPS3 that applied this study had higher expression stability than commonly used housekeeping genes. The application of rDNA-based primers in qPCR analysis was addressed.

Dataset Information

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

Publications

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

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