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Accurate gene expression analysis in Norway spruce ( Picea abies ) under diverse stress conditions requires the identification of stable reference genes for normalization. Notably, the literature lacks reports on suitable reference genes in Norway spruce. Here, we aimed to address this gap by identifying suitable reference genes for quantitative real-time PCR in Norway spruce across various stress conditions (drought, heat, pathogen infection) in seedlings, tissues (needle, phloem, root), and developmental stages (seedlings, mature trees). We evaluated the stability of 15 candidate reference genes and assessed their expression stability using five statistical algorithms (ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder). Our results highlight ubiquitin-protein ligase ( SP1 ), conserved oligomeric Golgi complex ( COG7 ), and tubby-like F-box protein ( TULP6 ) as the most stable reference genes, while succinate dehydrogenase ( SDH5 ) and heat shock protein 90 (HSP90) were the least stable under various experimental conditions . COG7 and TULP6 are novel candidate reference genes reported for the first time. The expression stability of the identified reference genes was further validated using dehydrin-like protein 5 ( PaDhn5 ) under drought conditions in Norway spruce. Pairwise variation analysis suggests that two reference genes were sufficient to normalize gene expression across all sample sets. This study provides a comprehensive analysis of reference gene stability under different experimental conditions and a catalog of genes for each condition, facilitating future functional genomic research in Norway spruce and related conifers.

Eustoma grandiflorum, commonly known as prairie gentian or Texas bluebells, is among the most popular agriculturally propagated species of cut flowers. Due to its widespread appeal, there is increasing interest in understanding the molecular genetic factors underlying floral development and resistance to abiotic stresses. We analyzed 18 potential reference genes in different organs, at different floral developmental stages and under drought- and salt-stress treatments, for use in RT-qPCR analysis. A total of four analytical tool packages, including geNorm, NormFinder, BestKeeper, and RefFinder were employed to determine the most appropriate reference genes under each treatment condition. The results demonstrate that different reference genes should be used for normalization under different experimental treatments. EgPP and EgPP2A2 were the most stable internal control genes across different organ types, EgPP and Eg18S were the most stable under salt-stress, EgPP and EgACT1 were the most stable across different floral development stages, and EgEF1A and EgTUA were the most stable reference genes under drought-stress. Additional gene expression analyses of EgMIXTA1, EgTOE1, and EgP5CS1 further confirmed the applicability of these reference genes. The results represent a significant contribution to future studies of reference gene selection for the normalization of gene expression in Eustoma grandiflorum.

The dynamic assembly and disassembly of microtubules (MTs) is essential for cell function. Although leaf senescence is a well-documented process, the role of the MT cytoskeleton during senescence in plants remains unknown. Here, we show that both natural leaf senescence and senescence of individually darkened Arabidopsis (Arabidopsis thaliana) leaves are accompanied by early degradation of the MT network in epidermis and mesophyll cells, whereas guard cells, which do not senesce, retain their MT network. Similarly, entirely darkened plants, which do not senesce, retain their MT network. While genes encoding the tubulin subunits and the bundling/stabilizing MT-associated proteins (MAPs) MAP65 and MAP70-1 were repressed in both natural senescence and dark-induced senescence, we found strong induction of the gene encoding the MT-destabilizing protein MAP18. However, induction of MAP18 gene expression was also observed in leaves from entirely darkened plants, showing that its expression is not sufficient to induce MT disassembly and is more likely to be part of a Ca²⁺-dependent signaling mechanism. Similarly, genes encoding the MT-severing protein katanin p60 and two of the four putative regulatory katanin p80s were repressed in the dark, but their expression did not correlate with degradation of the MT network during leaf senescence. Taken together, these results highlight the earliness of the degradation of the cortical MT array during leaf senescence and lead us to propose a model in which suppression of tubulin and MAP genes together with induction of MAP18 play key roles in MT disassembly during senescence.

Quinoa has been recognized as the sole “comprehensive nutritional crop”; however, it is susceptible to pre-harvest sprouting (PHS). While quantitative reverse transcription polymerase chain reaction (RT-qPCR) has been extensively employed for gene expression level detection, the selection of suitable reference genes is imperative to ensure precise gene expression quantification across diverse conditions. This study aims to identify stable reference genes in quinoa seeds under ABA and GA, in order to provide a basis for subsequent research on PHS. Seeds were subjected to different concentrations of ABA and GA (10 μM, 50 μM, 100 μM, and 200 μM). The most suitable treatment concentration was determined based on seed viability. Here, MON1, GAPDH, EIF3, EF1α, ACT, TUB1, and TUB6 were selected as candidate genes. The suitability of these reference genes under different conditions was assessed using various methods including Ct values, geNorm, NormFinder, BestKeeper, Delta Ct, and RefFinder. Based on the results obtained from the hormone experiments, it was observed that the application of 100 μM ABA and 200 μM GA yielded the most advantageous outcomes. Additionally, the most appropriate reference genes for different treatments are ACT and TUB1 (H2O treatment), EIF3 and MON1 (ABA, GA treatment and also for the combined data set of the three groups). However, GAPDH exhibited the least stability across all treatments. In summary, ACT is recommended as the reference gene for natural quinoa germination, while EIF3 and MON1 should be used for ABA and GA treatments. Resumo: A quinoa foi reconhecida como a única “cultura nutricional abrangente”; no entanto, é suscetível à germinação na pré-colheita (BHS). Embora a reação em cadeia da polimerase com transcrição reversa quantitativa (RT-qPCR) tenha sido amplamente empregada para detecção do nível de expressão gênica, a seleção de genes de referência adequados é essencial para garantir a quantificação precisa da expressão gênica em diversas condições. Este estudo tem como objetivo identificar genes de referência estáveis em sementes de quinoa tratadas com ABA e GA, a fim de fornecer uma base para pesquisas subsequentes em BPC. As sementes foram submetidas a diferentes concentrações de ABA e GA (10 μM, 50 μM, 100 μM e 200 μM). A concentração de tratamento mais adequada foi determinada com base na viabilidade das sementes. MON1, GAPDH, EIF3, EF1α, ACT, TUB1 e TUB6 foram selecionados como genes candidatos. A adequação destes genes de referência sob diferentes condições foi avaliada utilizando vários métodos, incluindo valores Ct, geNorm, NormFinder, BestKeeper, Delta Ct e RefFinder. Com base nos resultados obtidos nos experimentos com hormônios, observou-se que a aplicação de 100 μM de ABA e 200 μM de GA produziu os resultados mais vantajosos. Além disso, os genes de referência mais apropriados para diferentes tratamentos são ACT e TUB1 (tratamento com H2O), EIF3 e MON1 (tratamento com ABA, GA e para o conjunto de dados combinados dos três grupos). No entanto, o GAPDH exibiu a menor estabilidade em todos os tratamentos. Em resumo, o ACT é recomendado como gene de referência para a germinação natural da quinoa, enquanto o EIF3 e o MON1 devem ser utilizados para os tratamentos com ABA e GA.

Although many genes may serve as reference genes, they may cause different expression patterns by selecting different reference genes because no single gene is expressed consistently in all tested tissues of an organism under all environmental and developmental conditions. Thus, it is becoming increasingly important and necessary to identify suitable reference genes before performing gene expression analysis. Currently, there are several computational tools available for evaluating the stability of candidate reference genes. These tools are based on different statistical algorithms and may produce different rankings in stability within the same reference gene study. To date, the RefFinder is the only web-based tool available for comparing and evaluating housekeeping genes as candidates to be reference genes. In this tool, we integrated the four currently available computational programs (geNorm, NormFinder, BestKeeper, and the comparative ΔCt method) into a web-based tool for evaluating the stability and reliability of reference genes. According to the gene stability rankings derived from the four programs, we assigned an appropriate weight to each gene and calculated the geometric mean of weights for the final rankings. Aside from the overall ranking, a single program or combination of the four programs can be selected for evaluating the ranking of candidate reference genes. This tool has been widely used and validated by many research laboratories around the world. You may use this tool at http://www.heartcure.com.au/reffinder/  or  https://blooge.cn/RefFinder/ . You can also download this algorithm program from https://github.com/fulxie/RefFinder and setup on your own computer. RefFinder is developed by PHP. Users can deploy it to a Php-based server (Apache + PHP) and run it.

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.

The appropriate choice of reference genes is essential for accurate normalization of gene expression data obtained by the method of reverse transcription quantitative real-time PCR (RT-qPCR). In 2009, a guideline called the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) highlighted the importance of the selection and validation of more than one suitable reference gene for obtaining reliable RT-qPCR results. Herein, we searched the recent literature in order to identify the bacterial reference genes that have been most commonly validated in gene expression studies by RT-qPCR (in the first 5 years following publication of the MIQE guidelines). Through a combination of different search parameters with the text mining tool MedlineRanker, we identified 145 unique bacterial genes that were recently tested as candidate reference genes. Of these, 45 genes were experimentally validated and, in most of the cases, their expression stabilities were verified using the software tools geNorm and NormFinder. It is noteworthy that only 10 of these reference genes had been validated in two or more of the studies evaluated. An enrichment analysis using Gene Ontology classifications demonstrated that genes belonging to the functional categories of DNA Replication (GO: 0006260) and Transcription (GO: 0006351) rendered a proportionally higher number of validated reference genes. Three genes in the former functional class were also among the top five most stable genes identified through an analysis of gene expression data obtained from the Pathosystems Resource Integration Center. These results may provide a guideline for the initial selection of candidate reference genes for RT-qPCR studies in several different bacterial species.

miRNAs are small non-coding RNAs of about 18–25 nucleotides that negatively regulate gene expression at the post-transcriptional level. It was reported that a deregulation of their expression patterns correlates to the onset and progression of various diseases. Recently, these molecules have been identified in a great plethora of biological fluids, and have also been proposed as potential diagnostic and prognostic biomarkers. Actually, real time quantitative polymerase chain reaction is the most widely used approach for circulating miRNAs (c-miRNAs) expression profiling. Nevertheless, the debate on the choice of the most suitable endogenous reference genes for c-miRNAs expression levels normalization is still open. In this regard, numerous research groups are focusing their efforts upon identifying specific, highly stable, endogenous c-mRNAs. The aim of this review is to provide an overview on the reference genes currently used in the study of various pathologies, offering to researchers the opportunity to select the appropriate molecules for c-miRNA levels normalization, when their choosing is based upon literature data.

Reverse transcription quantitative real-time PCR (RT-qPCR) is a sensitive technique for quantifying gene expression, but its success depends on the stability of the reference gene(s) used for data normalization. Only a few studies on validation of reference genes have been conducted in fruit trees and none in banana yet. In the present work, 20 candidate reference genes were selected, and their expression stability in 144 banana samples were evaluated and analyzed using two algorithms, geNorm and NormFinder. The samples consisted of eight sample sets collected under different experimental conditions, including various tissues, developmental stages, postharvest ripening, stresses (chilling, high temperature, and pathogen), and hormone treatments. Our results showed that different suitable reference gene(s) or combination of reference genes for normalization should be selected depending on the experimental conditions. The RPS2 and UBQ2 genes were validated as the most suitable reference genes across all tested samples. More importantly, our data further showed that the widely used reference genes, ACT and GAPDH, were not the most suitable reference genes in many banana sample sets. In addition, the expression of MaEBF1, a gene of interest that plays an important role in regulating fruit ripening, under different experimental conditions was used to further confirm the validated reference genes. Taken together, our results provide guidelines for reference gene(s) selection under different experimental conditions and a foundation for more accurate and widespread use of RT-qPCR in banana.

The stability of internal reference genes is of great significance for the study of gene functions, and the development and screening of stable internal reference genes is the key to gene function analysis. Real-time reverse transcription quantitative PCR (qPCR) is an important tool to measure gene expression levels. Selection of stable reference genes for data normalization is a prerequisite. To date, the lack of studies on validation of reference genes in Ribes odoratum limits application of qPCR in Ribes odoratum . In this study, Through drought stress treatments and transcriptome analysis of Ribes odoratum , this study identified multiple genes with stable expression under drought stress. The expression stability of 17 candidate reference genes under drought stress was evaluated using the Genorm, NormFinder, BestKeeper, and Delta Ct algorithms. The results showed that Cluster-37.22228 , Cluster-37.22646 and Cluster-37.22897 were the top three stable reference genes, and the best reference genes that could be used as internal controls in Ribes odoratum was Cluster-37.22228 and Cluster-37.22646. These results provided for the first time a comprehensive list of stable reference genes for the normalization of qPCR analyses in Ribes odoratum under drought stress and will benefit the in-depth molecular biology research in the future.