Search Results (370)

Precision medicine approaches rely on accurate somatic variant detection, where the DNA input into genomic workflows is a key variable. However, there are no gold standard methods for total DNA quantification. In this study, a pentaplex reference gene panel using digital PCR (dPCR) was developed as a candidate reference method. The multiplex approach was compared between two assay chemistries, applied to healthy donor genomic DNA and plasma cell-free DNA (cfDNA) to measure the ERBB2 (HER2) copy number variation in cancer cell line DNA. The multiplex approach demonstrated robust performance with the two assay chemistries, demonstrating comparable results and a wide dynamic range. Ratios of reference genes were close to the expected 1:1 in healthy samples; however, some small but significant differences (<1.2-fold) were observed in one of the five targets. Expanded relative measurement uncertainty was 12.1–19.8% for healthy gDNA and 9.2–25.2% for cfDNA. The multiplex approach afforded lower measurement uncertainty compared to the use of a single reference for total DNA quantification, which is an advantage for its potential use as a calibration method. It avoided potential biases in the application to CNV quantification of cancer samples, where cancer genome instability may be prominent. Full article

The grassland caterpillar Gynaephora qinghaiensis (Lepidoptera: Lymantriidae) is a dominant pest species in the alpine meadows of the Tibetan Plateau. Elucidating changes in key gene expression patterns will provide molecular insights into the adaptive evolutionary mechanisms of insects. Quantitative real-time PCR (qRT-PCR) is currently the predominant analytical methodology for assessing gene expression levels. However, variability among samples can compromise result reliability. Thus, selecting stably expressed reference genes for target gene normalization under diverse scenarios is critical. To date, suitable reference genes for G. qinghaiensis under varying experimental conditions have remained unidentified. In this study, the transcriptome data of G. qinghaiensis were obtained using the RNA-seq technique, and 13 candidate reference genes were selected. Four independent algorithms—ΔCt, geNorm, NormFinder, and BestKeeper—as well as a comprehensive online platform, RefFinder, were employed to evaluate the stability under six experimental conditions (tissues, developmental stages, sexes, temperatures, starvation, and insecticide treatments). Our findings identified the following optimal reference gene combinations for each experimental condition: RPS18, RPS15, and RPL19 for tissue samples; RPL19, RPS15, and RPL17 across developmental stages; RPS18 and RPS15 for different sexes; RPS8 and EF1-α under varying temperature conditions; RPL17 and RPL15 during starvation; and RPL19 and RPL17 following insecticide treatments. To validate the feasibility of the reference genes, we examined the expression of the target gene HSP60 in different tissues and under different temperatures. Our results established essential reference standards for qRT-PCR with G. qinghaiensis samples, laying the foundation for precise gene expression quantification in the future. Full article

Argynnis hyperbius (Lepidoptera: Nymphalidae), as an important environmental indicator species, has shown a gradual decline in its species richness amid intensifying climate change and increasing environmental pressures. Screening for optimal reference genes is fundamental to studying their physiological and adaptive changes using RT-qPCR technology. In this study, 10 commonly used reference genes (ACT, α-TUB, AK, GAPDH, EF1α, BTF3, RPS3, RPL10, RPL32, and RPL27) were selected, and their expression stability under different developmental stages, genders, and temperature treatments was evaluated using the RefFinder website. The results indicated that selecting a pair of reference genes could achieve the most reliable normalization analysis under all experimental conditions. Specifically, AK and EF1α were the most stably expressed reference genes across different developmental stages; ACT and RPL32 showed the most stable expression in different adult sexes; and EF1α and RPL27 exhibited stable expression under different temperature treatments. Additionally, this study used EF1α and RPL32, the most stable reference genes from all results, to normalize and analyze the relative transcription levels of HSP90 under different temperatures, revealing significant differences between the 4 °C group and both the 26 °C and 37 °C groups. The findings of this study will significantly improve the reliability of RT-qPCR detection and lay a foundation for in-depth research on the gene expression, physiology, and biochemical mechanisms of A. hyperbius. Full article

HIV/AIDS remains a major global health challenge, with immune dysfunction, chronic inflammation, and comorbidities sustained by latent viral reservoirs that evade antiretroviral therapy. Euclea natalensis, a medicinal plant widely used in Southern African ethnomedicine, remains underexplored for its potential against HIV. An integrative systems pharmacology and molecular modeling framework was employed, including ADME profiling, target mapping, PPI network analysis, GO and KEGG pathway enrichment, BA-TAR-PATH analysis, molecular docking, MD simulations, and MM/GBSA calculations, to investigate the mechanistic roles of E. natalensis phytochemicals in HIV pathogenesis. Sixteen phytochemicals passed ADME screening and mapped to 313 intersecting host targets, yielding top ten hub genes with GO annotations in immune-metabolic, apoptotic, and nuclear signaling pathways. KEGG analysis revealed the enrichment of HIV-relevant pathways, including Th17 cell differentiation (hsa04659), PD-L1/PD-1 checkpoint (hsa05235), IL-17 signaling (hsa04657), HIF-1 signaling pathway (hsa04066), and PI3K-Akt (hsa04151). Lead phytochemicals, diospyrin and galpinone, strongly targeted key hub proteins (NFκβ1, STAT3, MTOR, HSP90AA1, and HSP90AB1), demonstrating favorable binding affinities, conformational stability, and binding free energetics compared to reference inhibitors. E. natalensis phytochemicals may modulate Th17 differentiation, HIV latency circuits, and comorbidity-linked signaling by targeting multiple host pathways, supporting their potential as multi-target therapeutic candidates for adjunct HIV/AIDS treatment and immunotherapy. Full article

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder caused by the BCR::ABL1 fusion gene, resulting from a reciprocal translocation between chromosomes 22 and 9. Quantification of BCR::ABL1 transcript levels in peripheral blood by RT-qPCR represents the gold standard for molecular response (MR) monitoring, providing essential clinical information on treatment efficacy. Xpert^® BCR-ABL Ultra is a fully automated in vitro diagnostic test that quantitatively detects e13a2 and e14a2 BCR::ABL1 transcripts using a single-use cartridge that integrates RNA extraction, cDNA synthesis, nested real-time PCR, and signal detection within a rapid, closed, and user-friendly system. In this study, we evaluated Xpert^® BCR-ABL Ultra as an alternative to validated systems currently used by four highly specialized Italian laboratories affiliated with the Italian national laboratory network for CML. A total of 129 peripheral blood samples from CML patients at various disease stages, along with two external quality control materials, were analyzed. We assessed the test’s repeatability, specificity, and stability. Concordance of BCR::ABL1%IS values generated by the different methods was evaluated using EUTOS criteria and Bland–Altman analysis. Finally, MR value concordance was analyzed based on European LeukemiaNet recommendations or calculated using the formula 2 − log₁₀(BCR::ABL1%IS). Xpert^® BCR-ABL Ultra demonstrated high repeatability and stability. The BCR::ABL1%IS values obtained with this assay showed strong concordance with those generated by local reference methods, and MR classifications were consistent across platforms. These findings confirm the robustness, accuracy, and efficiency of the Xpert^® BCR-ABL Ultra assay, supporting its use as a reliable alternative to currently validated systems for the routine clinical monitoring of CML patients. Full article

rDNA is abundant in various organisms, typically expressed as conserved tandem repeats. It plays a crucial role in ribosome synthesis, gene transcription, and expression, and it affects the occurrence of diseases in both animals and plants, aging, protein synthesis, genomic stability, and genome evolution across a wide range of organisms. Among the different types of rDNA, 35S rDNA (also referred to as 45S rDNA) and 5S rDNA are particularly important in plant research. The use of 35S rDNA and 5S rDNA as probes has enabled the study of chromosomal composition, revealing species characteristics that are valuable for crop breeding, evolutionary biology, systematics, and other fields. This review focuses on the application of 35S rDNA and 5S rDNA and discusses research findings on sugarcane and its related germplasm that have been obtained through fluorescence in situ hybridization. This information has provided a foundation for understanding the genetic relationships, genetics, breeding, and evolutionary classification of sugarcane. Full article

Background/Objectives: Optimal reference genes for normalizing RT-qPCR data depend on the species, treatments, developmental stages, and other conditions. Pastor roseus is a long-distance migratory bird with potential applications in locust biological control. This study applied reverse transcription quantitative PCR (RT-qPCR) to evaluate the expression stability of six genes (RPS2, ACTB, B2M, SDHA, UBE2G2, and RPL4) in blood samples from female, male, and nestling P. roseus. Methods: An integrated analysis of the expression stability of six reference genes was performed using three statistical algorithms: GeNorm, BestKeeper, and NormFinder. Results: The results showed that SDHA, ACTB, and B2M exhibited the highest expression stability among the candidate reference genes. The optimal number of reference genes was two, as determined by a pairwise variation analysis using GeNorm. Subsequent comprehensive validation using RefFinder identified SDHA/ACTB as the optimal reference gene pair for normalizing gene expression data for P. roseus. Conclusions: These findings establish a robust foundation for ensuring data accuracy in functional genomic studies of P. roseus. Full article

Accurate normalization of target gene expression in qRT-PCR experiments requires the development of stable and efficient housekeeping reference genes, particularly for tissue-specific genes in a given organ. Phyllanthus emblica L., an economically important cash tree, has been applied as medical or functional fruit for years in Asian countries due to its fruit that contains rich and diverse active compounds. Developing housekeeping reference genes is critical to investigate the physiological and molecular regulation of fruit development and ripening for P. emblica genetic improvement in breeding practice. Here, based on the expressional stabilities and efficiencies, expression profiles of 13 candidate genes at various fruit development stages were compared between two accessions using expression levels and multiple statistical methods, including BestKeeper, NormFinder, geNorm, ΔCt, and RefFinder. The validation test was conducted through qRT-PCR analysis of two fruit tissue-specific genes, PeGASA and PeMLP, across the different fruit development stages, combined with the comparison of gene expression consistency between qRT-PCR and transcriptomic data. These analyses identified PeACT and PeUBQ6, two regulators of tissue development and ripening, to be the most suitable housekeeping reference genes. Thus, we recommended PeACT and PeUBQ6 can serve as housekeeping reference genes for conducting qRT-PCR analysis in P. emblica fruit, helpful for investigating gene expression related to fruit development and ripening using the qRT-PCR technique. Full article

The harsh, cold, and dry winters in northern China necessitate burying wine grapevines (Vitis vinifera) for winter protection. In this study, we screened for stably expressed reference genes in wine grapes (V. vinifera) under low-temperature stress at 4 °C (chilling) and −15 °C (freezing). A cold-resistant line “Hanniang 1301” and its cold-sensitive parent ‘Cabernet Sauvignon’ were treated at 4 °C and −15 °C for varying durations. Nineteen candidate reference genes were selected for qPCR analysis. Gene stability under chilling and freezing stress was evaluated using the following five algorithms: Delta CT (ΔCt), geNorm, NormFinder, BestKeeper, and RefFinder. The optimal reference genes under chilling (4 °C) and freezing (−15 °C) conditions were pairs with dual-reference combinations. However, the genes selected differed between chilling and sub-freezing temperatures. For chilling stress (4 °C), EF1α-1 and EF1α-2 were the most stable. Meanwhile, for freezing stress (−15 °C), GAPDH and Actin were optimal. We identified suitable reference genes for gene expression studies in wine grapes under low-temperature stress; this establishes a theoretical foundation for optimizing reference gene selection in plants under other abiotic stresses. Full article

Propsilocerus akamusi (Tokunaga, 1938) larvae serve as key bioindicators for water quality assessment. This study identifies optimal reference genes for RT-qPCR under diverse experimental conditions. Fifteen candidate genes commonly employed in other insect species were selected, candidate genes commonly used in other insect species. Homologous genes were identified in the P. akamusi genome through sequence alignment with their Drosophila melanogaster counterparts. Expression stability across developmental stages, body parts, temperature variations, and deltamethrin/nickel chloride exposures was systematically evaluated using geNorm, NormFinder, BestKeeper, and ΔCt methods. RPL32 exhibited the highest expression stability across different body parts of adults under varying temperature conditions, and RPS11 and RPL8 showed the greatest stability across developmental stages and in larvae exposed to different temperatures. Furthermore, under nickel chloride and deltamethrin treatments, RPS11 and RPL8 maintained the highest expression stability. The results indicated that the expression stability of reference genes varied under different conditions. Among different body parts of adults, RPL32 and RPL4 exhibited the most stable expression. Across different developmental stages, RPS11 and RPL8 performed best in terms of expression stability. Under different temperature treatments, RPL32 and RPL4 remained stable in adults, while RPS11 and RPL8 showed the greatest stability in larvae. Similarly, under nickel chloride and deltamethrin treatments, RPS11 and RPL8 demonstrated the most stable expression in larvae. Through the above research, we can advance ecosystem-impact insights and bolster environmental protection and water quality monitoring. Full article

Dyspepsia or indigestion is known to be a symptom of several gastrointestinal issues including gastroesophageal reflux disease (GERD) and gastric cancer. With the ever-increasing popularity of traditional herbal medicines, the research selected rosemary, or Salvia rosmarinus, due to its historical ethnopharmacological applications and ease of cultivation. Potential targets of molecules from S. rosmarinus are explored for the molecular pathogenesis of functional dyspepsia. Through a network pharmacology approach, it has been shown that there is a significant interaction between the disease and the plant’s compounds. The pathways involving the target genes of S. rosmarinus that are related to functional dyspepsia were revealed to be implicated in the development of certain diseases like gastric cancer and fibrosis which are both precursors to dyspepsia. Moreover, through molecular docking, the results of the pathway analyses were computationally validated indicating that the compound luteolin has the most significant interaction against dyspepsia-related genes. It effectively promotes apoptosis in cancer pathways, reducing the chances of gastric cancer carcinogenesis. To further validate these findings, molecular dynamics simulation was performed to compare the stability and binding behavior of the top-performing compound against the reference compound. The results of this study could be a possible basis in developing pharmaceuticals against gastrointestinal diseases, specifically, dyspepsia. Full article

Accurate normalization is crucial for reliable gene expression quantification and depends on stably expressed housekeeping genes (HKGs) as internal controls. However, HKGs expression varies with developmental stage, tissue type, and treatments, potentially introducing bias and compromising data accuracy. Thus, validating candidate reference genes under defined conditions is essential. Reynoutria, also known as giant Asian knotweeds, is a Polygonaceae family genus of several medicinal plants producing a diverse array of specialized metabolites of pharmacological interest. Outside their native range, these plants are also noxious invasive weeds, causing significant environmental and economic threats. Research on stable reference genes in these species is limited, with a primary focus on R. japonica. To enable accurate gene expression analysis related to specialized metabolism and natural product biosynthesis, we aimed to identify the most stable reference genes across the most common species: R. japonica Houtt., R. sachalinensis (F. Schmidt) Nakai, and their hybrid—R. × bohemica Chrtek & Chrtková. In this study, we evaluated twelve candidate HKGs (ACT, TUA, TUB, GAPDH, EF-1γ, UBQ, UBC, 60SrRNA, eIF6A, SKD1, YLS8, and NDUFA13) across three tissue types (rhizomes, leaves, and flowers) from three Reynoutria species sampled at peak flowering. Primer specificity and amplification efficiency were confirmed through standard-curve analysis. We assessed expression stability using ΔCt, geNorm, NormFinder, and BestKeeper, and generated comprehensive rankings with RefFinder. Our integrated analysis revealed organ- and species-dependent stability differences, yet identified up to three reference genes suitable for interspecific normalization in Reynoutria. This represents the first systematic, comparative validation of HKGs across closely related knotweed species, providing a robust foundation for future transcriptomic and functional studies of their specialized metabolism and other biological processes. Full article

Background: Postbiotics with anti-adipogenic properties can significantly modify adipocyte metabolism by influencing key cellular pathways involved in lipid accumulation. In preliminary in vitro studies, it is essential to monitor various cellular and subcellular variables, including gene expression and protein synthesis potential, through RT-qPCR analysis. It is also crucial to select internal controls carefully and evaluate their stability for effective normalization and accurate interpretation of the results. Methods: In this study, we assessed the stability of six commonly used housekeeping genes: GAPDH, Actb, HPRT, HMBS, 18S, and 36B4. We analyzed their variability in mature 3T3-L1 adipocytes treated with supernatants from newly isolated Lacticaseibacillus paracasei strains. Our analysis combined classical statistical methods, a ∆Ct analysis, and software algorithms such as geNorm, NormFinder, BestKeeper, and RefFinder. Results: Our stepwise, multiparameter strategy for selecting reference genes led to the exclusion of Actb and 18S as the most variable reference genes. We identified HPRT as the most stable internal control. Additionally, HPRT and HMBS emerged as a stable pair, while the recommended triplet of genes for reliable normalization consists of HPRT, 36B4, and HMBS. Conclusions: The widely used putative genes in similar studies—GAPDH and Actb—did not confirm their presumed stability, which once again emphasizes the need for experimental validation of internal controls to increase the accuracy and reliability of gene expression. Combining a unique biological model—postbiotic-treated adipocytes—with multiple algorithms integrated into a single workflow allows us to provide a methodological template applicable to similar nutritional and metabolic research settings. Full article

Quinoa (Chenopodium quinoa), a stress-tolerant pseudocereal ideal for studying abiotic stress responses, was used to systematically identify optimal reference genes for qPCR normalization under gradient stresses: low temperatures (LT group: −2 °C to −10 °C), heat (HT group: 39° C to 45 °C), and drought (DR group: 7 to 13 days). Through multi-algorithm evaluation (GeNorm, NormFinder, BestKeeper, the ΔCt method, and RefFinder) of eleven candidates, condition-specific optimal genes were established as ACT16 (Actin), SAL92 (IT4 phosphatase-associated protein), SSU32 (Ssu72-like family protein), and TSB05 (Tryptophan synthase beta-subunit 2) for the LT group; ACT16 and NRP13 (Asparagine-rich protein) for the HT group; and ACT16, SKP27 (S-phase kinase), and NRP13 for the DR group, with ACT16, NRP13, WLIM96 (LIM domain-containing protein), SSU32, SKP27, SAL92, and UBC22 (ubiquitin-conjugating enzyme E2) demonstrating cross-stress stability (global group). DHDPS96 (dihydrodipicolinate synthase) and EF03 (translation elongation factor) showed minimal stability. Validation using stress-responsive markers—COR72 (LT), HSP44 (HT), COR413-PM (LT), and DREB12 (DR)—confirmed reliability; COR72 and COR413-PM exhibited oscillatory cold response patterns, HSP44 peaked at 43 °C before declining, and DREB12 showed progressive drought-induced upregulation. Crucially, normalization with unstable genes (DHDPS96 and EF03) distorted expression profiles. This work provides validated reference standards for quinoa transcriptomics under abiotic stresses. Full article

Estrogen receptor alpha (ERα) plays a vital role in the development and progression of breast cancer by regulating the expression of genes associated with cell proliferation in breast tissue. ERα inhibition is a key strategy in the prevention and treatment of breast cancer. Previous research modified chalcone compounds into pyrazoline benzenesulfonamide derivatives (Modifina) which show activity as an ERα inhibitor. This study aimed to design novel pyrazoline benzenesulfonamide derivatives (PBDs) as ERα antagonists using in silico approaches. Structure-based and ligand-based drug design approaches were used to create drug target molecules. A total of forty-five target molecules were initially designed and screened for drug likeness (Lipinski’s rule of five), cytotoxicity, pharmacokinetics and toxicity using a web-based prediction tools. Promising candidates were subjected to molecular docking using AutoDock 4.2.6 to evaluate their binding interaction with ERα, followed by molecular dynamics simulations using AMBER20 to assess complex stability. A pharmacophore model was also generated using LigandScout 4.4.3 Advanced. The molecular docking results identified PBD-17 and PBD-20 as the most promising compounds, with binding free energies (ΔG) of −11.21 kcal/mol and −11.15 kcal/mol, respectively. Both formed hydrogen bonds with key ERα residues ARG394, GLU353, and LEU387. MM-PBSA further supported these findings, with binding energies of −58.23 kJ/mol for PDB-17 and −139.46 kJ/mol for PDB-20, compared to −145.31 kJ/mol, for the reference compound, 4-OHT. Although slightly less favorable than 4-OHT, PBD-20 demonstrated a more stable interaction with ERα than PBD-17. Furthermore, pharmacophore screening showed that both PBD-17 and PBD-20 aligned well with the generated model, each achieving a match score of 45.20. These findings suggest that PBD-17 and PBD-20 are promising lead compounds for the development of a potent ERα inhibitor in breast cancer therapy. Full article