Search Results (215)

Asia minor bluegrass (Polypogon fugax), a widespread Poaceae weed, exhibits broad tolerance to abiotic stresses. Validated reference genes (RGs) for reliable RT-qPCR normalization in this ecologically and agriculturally significant species remain unidentified. This study identified eight candidate RGs using transcriptome data from seedling tissues. We assessed the expression stability of these eight RGs across various abiotic stresses and developmental stages using Delta Ct, BestKeeper, geNorm, and NormFinder algorithms. A comprehensive stability ranking was generated using RefFinder, with validation performed using the target genes COR413 and P5CS. Results identified EIF4A and TUB as the optimal RG combination for normalizing gene expression during heat stress, cold stress, and growth stages. EIF4A and ACT were most stable under drought stress, EIF4A and 28S under salt stress, and EIF4A and EF-1 under cadmium (Cd) stress. Furthermore, EIF4A and UBQ demonstrated optimal stability under herbicide stress. Additionally, application of validated RGs revealed higher acetyl-CoA carboxylase gene (ACCase) expression in one herbicide-resistant population, suggesting target-site gene overexpression contributes to resistance. This work presents the first systematic evaluation of RGs in P. fugax. The identified stable RGs provide essential tools for future gene expression studies on growth and abiotic stress responses in this species, facilitating deeper insights into the molecular basis of its weediness and adaptability. Full article

Accurate gene expression quantification using reverse transcription quantitative PCR (RT-qPCR) requires stable reference genes (RGs) for reliable normalization. However, few studies have systematically identified RGs suitable for simultaneous high salt, alkaline, and high-temperature conditions. This study addresses this gap by evaluating the stability of eight candidate RGs in the anaerobic halophilic alkalithermophile Natranaerobius thermophilus JW/NM-WN-LF^T under combined salt, alkali, and thermal stresses. The stability of these candidate RGs was assessed using five statistical algorithms: Delta CT, geNorm, NormFinder, BestKeeper, and RefFinder. Results indicated that recA exhibited the highest expression stability across all tested conditions and proved adequate as a single RG for normalization in both RT-qPCR and droplet digital PCR (ddPCR) assays. Furthermore, recA alone or combined with other RGs (sigA, rsmH) effectively normalized the expression of seven stress-response genes (proX, opuAC, mnhE, nhaC, trkH, ducA, and pimT). This work represents the first systematic validation of RGs under polyextreme stress conditions, providing essential guidelines for future gene expression studies in extreme environments and aiding research on microbial adaptation mechanisms in halophilic, alkaliphilic, and thermophilic microorganisms. Full article

Immunotherapy has emerged as a key modality in cancer treatment, yet its effectiveness varies significantly among patients, often due to the metabolic stress imposed by the tumor microenvironment. Hypoxia, a major factor in the tumor microenvironment, results from the high metabolic rate of tumor cells and inadequate vascularization, impairing immune cells’ function and potentially influencing gene expression profiles. Despite the widespread use of quantitative real-time PCR in immunological studies, to the best of our knowledge, data on reference gene stability in human peripheral blood mononuclear cells under hypoxic conditions is limited. In our study, we assessed the expression stability of commonly used reference genes (S18, HPRT, IPO8, RPL13A, SDHA, PPIA, and UBE2D2) in both non-stimulated and CD3/CD28-activated peripheral blood mononuclear cells cultured under normoxic, hypoxic (1% O₂), and chemically induced hypoxic conditions for 24 h. Analysis using four different algorithms—delta Ct, geNorm, NormFinder, and BestKeeper—identified RPL13A, S18, and SDHA as the most suitable reference genes for human peripheral blood mononuclear cells under hypoxic conditions. In contrast, IPO8 and PPIA were found to be the least suitable housekeeping genes. The study provides essential insights into the stability of reference genes in peripheral blood mononuclear cells under hypoxic conditions, a critical but understudied aspect of immunological research. Given the significant impact of hypoxia on T cell metabolism and function in the tumor microenvironment, selecting reliable reference genes is crucial for accurate gene expression analysis. Our findings will be valuable for future studies investigating hypoxia-driven metabolic reprogramming in immune cells, ultimately contributing to a better understanding of T cell responses in cancer immunotherapy. Full article

Xanthomonas phaseoli pv. dieffenbachiae (Xpd), the causal agent of bacterial blight in Anthurium within the Araceae family, is listed as an EPPO A2 quarantine organism. Although the whole genome of Xpd has been sequenced, the molecular mechanisms underlying anthurium bacterial blight (ABB) remain unknown. Selecting an optimal reference gene is crucial for obtaining accurate and reliable gene expression profiles during the initial interactions between Xpd and Anthurium. The stability of four reference genes was evaluated by applying three statistical methods—BestKeeper, geNorm, and delta Ct (ΔCt)—using reverse-transcription quantitative PCR (RT-qPCR) data. The rpoD and gyrB genes exhibited the most consistent gene expression profiles, whereas atpD and thyA were less stable at four time points (0, 0.5, 1, and 2 h) during the interactions between Xpd and susceptible A. andreanum cultivar ‘Marian Seefurth.’ The suitability of these reference gene candidates was validated by normalizing the gene expression levels of four pathogenicity-related genes. The highly upregulated expression of gumD, which encodes xanthan biosynthesis glycosyltransferase, observed after 1 h of interaction, suggests it may be a key virulence determinant in the Xpd–Anthurium pathosystem. The stable reference genes identified here will facilitate more accurate and comprehensive gene expression studies in the Xpd–Anthurium pathosystem going forward. Full article

Background: Xanthomonas oryzae pv. oryzae (Xoo) causes rice leaf blight (BLB) and poses a major threat to global rice production. In rice production, tetramycin agents provide good control of rice leaf blight, while the standardization of the reference genes of Xoo under tetramycin stress has not been reported. The aim of this study was to identify the most stable reference genes for quantitative PCR analysis of Xoo under tetramycin stress. Methods: Six candidate reference genes, gyrB (RNA polymerase β gene), GADPH (glyceraldehyde-3-phosphate dehydrogenase gene), recA (recombinase A gene), gyrA (citrate synthase encoding gene), dnaK (molecular chaperone protein gene), and 16S rRNA (16S ribosomal RNA gene) were selected and their expression stability was assessed under tetramycin stress conditions using real-time quantitative PCR (qRT-PCR). GeNorm, NormFinder, BestKeeper and RefFinder were used to assess the expression stability, the relative expression values of the eight genes involved QS (Quorum sensing) pathway under tetramycin stress were used to validate by the rpf (regulation of pathogenic factors) gene family. Results: 16S rRNA expression was most stable under tetracycline stress, whereas GADPH was the least. The rpf gene family showed a highly stable expression level, confirming the reliability of 16S r RNA as a reference gene in the study of Xoo under tetramycin stress. Conclusions: 16S rRNA was identified as the best reference gene for Xoo gene expression analysis under tetramycin stress. It provides a reliable support for the molecular research on the control strategy of rice BLB. Full article

Plant abiotic stresses are the main cause of significant crop losses worldwide. The moss Pseudocrossidium replicatum is highly tolerant to different types of abiotic stress, such as desiccation. Our group is interested in identifying and characterising differentially expressed genes in response to abiotic stress in this species. However, a collection of validated reference genes for RT-qPCR analysis is essential to normalise the expression of genes in response to the conditions of interest. Here, we assessed 13 candidate reference genes for P. replicatum based on their expression stability across transcriptomes from six abiotic stress-related conditions using the RefFinder, BestKeeper, geNorm, and NormFinder programs. The stability and reliability of the proposed reference genes were evaluated under six experimental conditions: control, dehydration, rehydration, abscisic acid (ABA), NaCl, and sorbitol. Interestingly, most proposed reference genes exhibited high stability (low M values) across all analysed abiotic stress conditions. A pairwise variation analysis indicated that only one pair is necessary to normalise RT-qPCR experiments. Each gene was confirmed to normalise the expression of both upregulated and downregulated genes. This represents the first report of validated reference genes for RT-qPCR gene expression studies under abiotic stress in the protonemal tissue of a fully desiccation-tolerant moss. Full article

The clover cutworm, Scotogramma trifolii Rottemberg (Lepidoptera: Noctuidae), is a globally distributed polyphagous pest causing significant economic losses to agricultural crops. RT-qPCR is a gold-standard technique for gene expression analysis, yet its accuracy depends critically on stable reference genes for data normalization. To address the lack of validated reference genes in S. trifolii, we evaluated six candidate genes (β-actin, RPL9, GAPDH, RPL10, EF1-α, and TUB) across four developmental stages (egg, larva, pupa, and adult) and six adult tissues (head, thorax, abdomen, wings, legs, and antennae) using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. Stability analysis identified β-actin, RPL9, and GAPDH as the most reliable reference genes for developmental stage normalization, while RPL10, GAPDH, and TUB were validated for adult tissues. Functional validation using the odorant receptor gene StriOR20 revealed significant discrepancies in relative expression levels when normalized with unstable reference genes (TUB and RPL9), emphasizing the necessity of rigorous reference gene selection. This study establishes the first comprehensive reference gene panel for S. trifolii, providing a robust foundation for gene expression studies in this agriculturally important pest. Full article

The selection of appropriate reference genes is critical for standardizing quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) data, thereby ensuring accurate and reliable results of gene expression analysis. In this study, we identified 10 candidate reference genes (encoding α-tubulin, G6PDH, Rab1, RT, RPS13, β-tubulin, DPH1, HSP90, GAPDH, and CP) and evaluated their suitability for use as reference genes in the pest insect, Bactrocera tau. Analysis was conducted using three software-based methods—Delta CT, NormFinder, and BestKeeper—alongside the online tool RefFinder. Expression levels of these genes were analyzed across various B. tau developmental stages and body parts. The overall ranking of reference gene stability scores was as follows: α-tubulin > G6PDH > CP > β-tubulin > RT > HSP90 > GAPDH > DPH1 > RPS13 > Rab1. Ultimately, α-tubulin and G6PDH were identified as the most stable reference genes for B. tau. Full article

Selecting the right reference genes for data normalization is the only way to ensure the precision and reproducibility of gene expression measurement using qRT-PCR. Ottelia cordata is a member of the Hydrocharitaceae family in aquatic plants that exhibits both floating and submerged leaf forms. It has recently drawn interest as a possible model plant for research into non-KRANZ C₄ photosynthesis and heteroblastic leaves. Our earlier research has demonstrated bias in gene expression analysis when actin or GAPDH, two common reference genes, are used for normalization. Furthermore, there has been no study on the Hydrocharitaceae family reference gene selection published to date. To standardize qRT-PCR in O. cordata, seven genes were chosen from a transcriptome database: ACT7, EF1_α, GAPDH, BRCC36, PP2A, UBC7, and UBQ. We conducted qRT-PCR experiments in various tissues, leaves in different developmental stages, leaves in high/low carbon treatment, and leaves in high/low temperature treatment. After analyzing the stability using five statistical methods (geNorm, normFinder, comparative ΔCt, bestKeeper, and comprehensive analysis), PP2A and UBQ were identified as the most stable genes. BRCC36 was identified as a new reference gene in plants. Finally, by contrasting the expression patterns of pepc2, a crucial gene connected to C₄ photosynthesis, in floating and submerged leaves, PP2A, UBQ, and UBC7 were verified. Of these, PP2A and UBQ were shown to be the superior gene for the precise qRT-PCR data normalization. The results of this study offer the initial information concerning reference gene identification for O. cordata as well as the first data in Hydrocharitaceae plants. It will make it easier to do more gene function and molecular biology research on O. cordata and other closely related species. Full article

Clinacanthus nutans is a valuable traditional medicinal plant that contains enriched active compounds such as triterpenoids and flavonoids. Understanding the accuulation process of these secondary metabolites in C. nutans requires exploring gene expression regulation under abiotic stresses and hormonal stimuli. qRT-PCR is a powerful method for gene expression analysis, with the selection of suitable reference genes being paramount. However, reports on stably expressed reference genes in C. nutans and even across the entire family Acanthaceae are limited. In this study, we evaluated the expression stability of 12 candidate reference genes (CnUBQ, CnRPL, CnRPS, CnPTB1, CnTIP41, CnACT, CnUBC, CnGAPDH, Cn18S, CnCYP, CnEF1α, and CnTUB) in C. nutans across different tissues and under abiotic stresses and MeJA treatment using three programs (geNorm, NormFinder, and BestKeeper). The integrated ranking results indicated that CnUBC, CnRPL, and CnCYP were the most stably expressed genes across different tissues. Under abiotic stress conditions, CnUBC, CnRPL, and CnEF1α were the most stable, while under MeJA treatment, CnRPL, CnEF1α, and CnGAPDH exhibited the highest stability. Additionally, CnRPL, CnUBC, and CnEF1α were the most stable reference genes across all tested samples, whereas CnGAPDH was the least stable. CnRPL, consistently ranking among the top three most stable genes, may therefore serve as an ideal reference gene for qRT-PCR analysis in C. nutans. To further validate the selected reference genes, we assessed the expression of two key biosynthetic genes, CnPAL and CnHMGR. The results confirmed that using the most stable reference genes yielded expression patterns consistent with biological expectations, while using unstable reference genes led to significant deviations. These findings offer valuable insights for accurately quantifying target genes via qRT-PCR in C. nutans, facilitating investigations into the mechanisms underlying active compound accumulation. Full article

S. pupariae is a newly discovered species of parasitoid wasps. Research into its development, behavioral genetics, and molecular mechanisms provides valuable insights for improving integrated pest management strategies. Quantitative real-time PCR (qRT-PCR) is the most commonly used method for analyzing gene expression. This method requires the identification of stable reference genes to accurately evaluate transcriptional level variations. In this study, eight candidate reference genes (TUB, TBP, RPS18, GAPDH, 18S rRNA, RPL32, Actin, and EF1-α) were identified and evaluated for their suitability as reference genes. Gene expression levels across different developmental stages were analyzed using three software tools, GeNorm, NormFinder, and BestKeeper, and the online tool RefFinder. The overall ranking of reference gene stability was as follows: RPS18 > 18S rRNA > RPL32 > GAPDH > Actin > TUB > TPB > EF1-α. Ultimately, RPS18 was determined to be the most stable reference gene. Full article

To identify stable reference genes for qRT-PCR analysis across different developmental stages and color variations of tender leaves in Litsea coreana, seven candidate reference genes were selected based on existing transcriptome data. qRT-PCR was performed on tender leaves of L. coreana at various stages and under different color conditions. The stability of these genes was evaluated using GeNorm (version 2003), NormFinder (version 0953), BestKeeper (version 2003), and ReFinder software (version 2004). The most stable genes were selected, and the stability of the chosen reference genes was validated. RPL and ACT were the most stable genes across different leaf developmental stages, while ACT and EF1-α showed the highest stability across different leaf colors. Overall, ACT and EF1-α were the most stable reference genes for both developmental stages and color variations. ACT and EF1-α can be used as reliable reference genes for gene expression studies in the color change process of L. coreana tender leaves. This will provide a foundation for further research into the molecular mechanisms of leaf color changes and the development of color regulation genes in L. coreana. Full article

Bacillus cereus is a Gram-positive pathogen associated with foodborne illnesses and severe non-gastrointestinal infections. Robust tools for accurate gene transcription analysis are essential for studying toxin gene expression dynamics and deciphering the complex regulatory networks orchestrating the expression of toxin and virulence factors. This study aimed to identify reliable reference genes for normalizing reverse transcription quantitative PCR (RT-qPCR) data in toxigenic B. cereus. An emetic and an enteropathogenic strain were used as model organisms to establish a suitable reference gene set to monitor the dynamics of toxin gene transcription. Ten candidate reference genes were evaluated for their expression stability using geNorm, NormFinder, BestKeeper and the ΔCq method, with the final rankings integrated via RefFinder. Among the tested genes, rho, rpoD and recA were identified as the most stable expressed reference genes across all tested conditions. As shown in this proof-of-principle study, the established reference gene set provides a suitable tool to investigate the influence of extrinsic and intrinsic factors on toxin gene transcription. In conclusion, our newly established reference gene set provides a robust basis for studying toxin gene expression in B. cereus and contributes to a better understanding of its pathogenicity and potential strategies to mitigate its harmful effects. Full article

Background: Hypoxia triggers stress, leading to significant alterations in gene expression patterns, which in turn affect fish’s growth and development. Real-time quantitative PCR (RT-qPCR) is a pivotal technique for assessing changes in gene expression. However, its accuracy is highly contingent upon the stable expression of reference genes. Ribosomal RNA (18s), β-actin (actb), elongation factor 1-α (ef1a), α tubulin (tuba), and ribosomal protein L17 (rpl17) are the widely used reference genes, but their expression stability in the tissues of black rockfish under hypoxic conditions remains unclear. Methods: The expression of genes was detected by RT-qPCR and the stability was assessed by Delta Ct, geNorm, NormFinder, and BestKeeper algorithms. Results: Results showed that tuba exhibited stable expression in liver, heart, gill tissues under normoxic conditions, and in the liver and head kidney under hypoxic conditions. Ef1a was identified as the most stably expressed gene in gill tissue under hypoxia. For hypoxic heart studies, rpl17 and tuba were recommended as reference genes. 18s showed high stability in spleen tissue under hypoxic conditions. Actb was the most stably expressed gene in spleen and head kidney tissues under normoxic conditions. Conclusions: The identified reference genes exhibited tissue-specific stability, and it was necessary to select appropriate reference genes based on the specific tissue type for gene expression studies under hypoxic conditions. These findings help in enhancing the accuracy of gene expression analysis in the mechanism of hypoxia for black rockfish. Full article

Quantitative real-time PCR (qRT-PCR) is an essential tool for analyzing and selecting stable reference genes. In order to screen for suitable reference genes under high-temperature stress conditions in Arabidopsis, this study measured the relative expression levels of 17 candidate reference genes using qRT-PCR. Among these, four are traditional reference genes, while the remaining thirteen are candidate reference genes with no previous reports on their use as reference genes. The expression stability of the candidate reference gene expression was analyzed and evaluated using five methods: ΔCt, geNorm, NormFinder, BestKeeper, and RefFinder. The results indicated that the LHCB4.1 and LHCB5 genes displayed the highest stability in expression under high-temperature stress conditions. To verify the stability of the reference genes, we treated Arabidopsis with high-temperature stress, used the selected LHCB4.1 and LHCB5 as references, and analyzed the expression of the heat-responsive gene HSFA2 using qRT-PCR. The results showed that when LHCB4.1 and LHCB5 were used individually or in combination as reference genes, the relative expression of HSFA2 significantly increased and remained consistent under high-temperature treatment. This indicates that both LHCB4.1 and LHCB5 are suitable reference genes for qRT-PCR analysis in Arabidopsis exposed to high-temperature stress. The novel reference genes identified in this study will serve as a reliable reference standard for gene expression studies in Arabidopsis under high-temperature stress, thereby enhancing the accuracy and comparability of experimental data. Full article