Evaluation of reference genes for reverse transcription quantitative real-time PCR (RT-qPCR) studies in Silene vulgaris considering the method of cDNA preparation

Koloušková, Pavla; Stone, James D.; Štorchová, Helena

doi:10.1371/journal.pone.0183470

Cited by 6 publications

(7 citation statements)

References 72 publications

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“…The quantification of gene expression is of increasing importance in many different fields of plant biology. One of the condition for accurate gene expression measurements is based on the choice of housekeeping gene, as many of them fluctuates in response to various stresses, circadian rhythms, or developmental stage (Koloušková et al 2017). Reference genes are usually involved basic functions in cells and are expressed at relatively constant rates under different experimental conditions and across different organs.…”

Section: Discussionmentioning

confidence: 99%

Variability in expression profiles of Betulaceae spring pollen allergens in Central Europe region

et al. 2021

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Pollen allergies are an important and relevant question in the personal life of millions of people. They are well characterized on the genomic and proteomic level of allergens per se and on medicinal level, but the very few information exists about the natural variability and background of pollen allergen transcription patterns. Here, the variability of expression of Bet v 1, Bet v 2, Cor a 1 and Cor a 2 were performed. Both of the analysed species, Betulla verrucosa as well as Corylus avellana are clinically relevant in spring pollinosis in the area of central Europe. Bet v 1 and Bet v 2 expressions were subjected to compare in pollen for a total of twelve accessions from different areas. Cor a 1 and Cor a 2 expressions were subjected to comparison in pollen for a total of thirty-one natural pollen germplasm accessions. Expression changes of Bet v 1 were changed in three different mannersin six pollen samples was the expression higher than 1.5, three pollen samples have no relevant change and in three pollen samples was the expression lower about 0.5 times. Expression changes of Bet v 2 were as follows -five of the analysed samples were expressed more than 3 times when compared to the calibrator and in all of the other samples was the expression change in the difference that was not above 0.7 times. Expression changes of Cor a 1 were very similar among themselves and only in five samples was expression relevantly higher with at least 1.1 fold change. Expression changes of Cor a 2 were very similar among themselves and only in four samples was expression relevantly higher with at least 1.4 fold change.

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Section: Discussionmentioning

confidence: 99%

Variability in expression profiles of Betulaceae spring pollen allergens in Central Europe region

et al. 2021

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“…Ten candidate genes ( 18S rRNA , actin , ARF , COX , CYP , EF1α , GAPDH , H3 , RPL2 , and TUBα ) were selected for this study according to their homologous gene stability in other plant species, such as sweet potato ( Ipomoea batatas ) [ 38 ], radish ( Raphanus sativus ) [ 57 ], potato ( Solanum tuberosum ) [ 39 ], soybean ( Glycine max ) [ 40 ], kenaf ( Hibiscus cannabinus ) [ 34 ], corn poppy ( Papaver rhoeas ) [ 37 ], bladder campion ( Silene vulgaris ) [ 36 ], Achyranthes bidentata [ 35 ], and Baphicacanthus cusia [ 33 ]. The sequences of these candidate homologous genes in spinach were obtained from SpinachBase ( http://www.spinachbase.org ) [ 14 , 58 , 59 ].…”

Section: Methodsmentioning

confidence: 99%

Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress

Xie

Chang

et al. 2021

BioMed Research International

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Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is an accurate and convenient method for mRNA quantification. Selection of optimal reference gene(s) is an important step in RT-qPCR experiments. However, the stability of housekeeping genes in spinach (Spinacia oleracea) under various abiotic stresses is unclear. Evaluating the stability of candidate genes and determining the optimal gene(s) for normalization of gene expression in spinach are necessary to investigate the gene expression patterns during development and stress response. In this study, ten housekeeping genes, 18S ribosomal RNA (18S rRNA), actin, ADP ribosylation factor (ARF), cytochrome c oxidase subunit 5C (COX), cyclophilin (CYP), elongation factor 1-alpha (EF1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (H3), 50S ribosomal protein L2 (RPL2), and tubulin alpha chain (TUBα) from spinach, were selected as candidates in roots, stems, leaves, flowers, and seedlings in response to high temperature, CdCl2, NaCl, NaHCO3, and Na2CO3 stresses. The expression of these genes was quantified by RT-qPCR and evaluated by NormFinder, BestKeeper, and geNorm. 18S rRNA, actin, ARF, COX, CYP, EF1α, GAPDH, H3, and RPL2 were detected as optimal reference genes for gene expression analysis of different organs and stress responses. The results were further confirmed by the expression pattern normalized with different reference genes of two heat-responsive genes. Here, we optimized the detection method of the gene expression pattern in spinach. Our results provide the optimal candidate reference genes which were crucial for RT-qPCR analysis.

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“…A calibrator (the same cDNA sample present in all plates) was used to correct for run-to-run variation. Expression values were normalized against the reference gene encoding actin ( SvAC T), as its stable expression in S. vulgaris was reported by [ 40 ].…”

Section: Methodsmentioning

confidence: 99%

Differentially Expressed Genes Shared by Two Distinct Cytoplasmic Male Sterility (CMS) Types of Silene vulgaris Suggest the Importance of Oxidative Stress in Pollen Abortion

Krüger

Abeyawardana

Krüger

et al. 2020

Cells

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Cytoplasmic male sterility (CMS), encoded by the interacting mitochondrial and nuclear genes, causes pollen abortion or non-viability. CMS is widely used in agriculture and extensively studied in crops. Much less is known about CMS in wild species. We performed a comparative transcriptomic analysis of male sterile and fertile individuals of Silene vulgaris, a model plant for the study of gynodioecy, to reveal the genes responsible for pollen abortion in this species. We used RNA-seq datasets previously employed for the analysis of mitochondrial and plastid transcriptomes of female and hermaphrodite flower buds, making it possible to compare the transcriptomes derived from three genomes in the same RNA specimen. We assembled de novo transcriptomes for two haplotypes of S. vulgaris and identified differentially expressed genes between the females and hermaphrodites, associated with stress response or pollen development. The gene for alternative oxidase was downregulated in females. The genetic pathways controlling CMS in S. vulgaris are similar to those in crops. The high number of the differentially expressed nuclear genes contrasts with the uniformity of organellar transcriptomes across genders, which suggests these pathways are evolutionarily conserved and that selective mechanisms may shield organellar transcription against changes in the cytoplasmic transcriptome.

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Evaluation of reference genes for reverse transcription quantitative real-time PCR (RT-qPCR) studies in Silene vulgaris considering the method of cDNA preparation

Cited by 6 publications

References 72 publications

Variability in expression profiles of Betulaceae spring pollen allergens in Central Europe region

Variability in expression profiles of Betulaceae spring pollen allergens in Central Europe region

Selection and Validation of Reference Genes for RT-qPCR Analysis in Spinacia oleracea under Abiotic Stress

Differentially Expressed Genes Shared by Two Distinct Cytoplasmic Male Sterility (CMS) Types of Silene vulgaris Suggest the Importance of Oxidative Stress in Pollen Abortion

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