Search Results (225)

The present study evaluated the stability of candidate reference genes during adipogenic differentiation of 3T3-L1 cells cultured on different extracellular matrices. The aim was to investigate the effects of matrix composition and differentiation stage on the expression of candidate housekeeping genes and to compare validation strategies in dynamic in vitro models. Eleven candidate reference genes (18S, Actb, B2m, Gapdh, Hmbs, Hprt, Nono, Ppia, Rplp0, Tbp, and Ywhaz) were analyzed by RT-qPCR in 3T3-L1 cells cultured on TC, collagen, gelatin, and Matrigel at Days 7 and 14 of differentiation. Gene stability was assessed using geNorm, NormFinder, RefFinder, comparative ΔCt, BestKeeper, generalized linear model (GLM), linear mixed model (LMM), and correlation analyses with the adipogenic markers Pparg and Fasn. The results demonstrated that the expression of most housekeeping genes was influenced by matrix composition, differentiation stage, or their interaction. Actb and 18S exhibited the strongest condition-dependent variability and pronounced matrix sensitivity. Gapdh and Hprt showed significant correlations with both Pparg and Fasn, while Hmbs correlated with Fasn, suggesting that these reference genes may not be fully independent of adipogenic status. Ppia demonstrated markedly contrasting rankings across analytical approaches, highlighting limitations of single-method stability assessment. The findings confirm that universal housekeeping genes are unlikely to exist across different matrix conditions and differentiation stages. The results highlight the need for multi-level validation strategies and experimentally validated normalization panels to minimize normalization bias and avoid misleading RT-qPCR expression profiles. Functional validation identified B2m and Rplp0 as the most suitable two-gene normalization panel for the experimental model evaluated, whereas Tbp remained a strong complementary reference gene candidate. Full article

(1) Reverse transcription quantitative real-time PCR (RT-qPCR) requires reliable reference genes for accurate data normalization; however, optimal reference genes for the economically and ecologically valuable timber species Phoebe zhennan remain uncharacterized; (2) Here, we selected nine candidate reference genes derived from full-length transcriptome sequencing to evaluate their expression stability across abiotic (drought) and biotic (Colletotrichum fructicola infection) stresses. Transcript abundance was analyzed via RT-qPCR using four distinct algorithms (Delta Ct, geNorm, NormFinder, and BestKeeper), with RefFinder used to reconcile analytical discrepancies and generate a definitive consensus ranking; (3) Our analysis showed that expression stability is highly context-dependent: CYP20-1 and HSP70-1 were the most stable reference genes under drought stress, whereas Actin-101 and Actin constituted the optimal pair under disease stress. For cross-condition assessments, Actin-101 and β-Tubulin served as the most reliable baseline combination. Subsequent empirical validation quantifying stress-responsive transcripts demonstrated a significant positive correlation between RT-qPCR relative expression and corresponding RNA-seq data (drought: R = 0.80; disease: R = 0.76); (4) This study identifies and validates the first set of reference genes for P. zhennan, providing a foundation for accurate gene expression analysis in this species, which is crucial for understanding its response to environmental stresses. Full article

Diaphorencyrtus aligarhensis parasitizes the Asian citrus psyllid (ACP), Diaphorina citri, the primary insect vector responsible for transmitting Huanglongbing (HLB), a severe citrus disease. Screening of appropriate reference genes is a critical prerequisite for reliable RT-qPCR analysis, which is essential for investigating the functions of target genes in D. aligarhensis across diverse experimental conditions. However, to date, no validated reference genes have been reported for this species. This study assessed seven housekeeping genes in D. aligarhensis under six conditions (developmental stage, body tissue, population, temperature, diet, and starvation) using five stability algorithms (geNorm, BestKeeper, NormFinder, RefFinder, and ∆Ct). The results identified the most suitable reference genes for specific experimental conditions: EIF5A and RPL32 for the developmental stage; RPL13 and H3 for population comparisons; RPS6 and GAPDH for different feeding diets; RPL32 and RPS6 for starvation; RPL7A and RPS6 for different body tissues (head, thorax, abdomen) and temperature gradients (5 °C, 15 °C, 25 °C, 35 °C). Furthermore, the expression profiles of HSP70 were markedly different when normalized to the most versus the least stable reference genes across body tissues, diets, starvation durations, and temperatures. Our findings establish the first set of RT-qPCR reference genes for D. aligarhensis, providing a useful foundation for functional genomics research on this biological control agent. Full article

Reliable normalization is essential for accurate quantitative real-time PCR (qRT-PCR) analysis, yet suitable reference genes have not been systematically evaluated in Mugilogobius chulae, an emerging marine experimental fish used in physiological and environmental research. In this study, 17 candidate reference genes were evaluated in five tissues (brain, gill, gonad, intestine, and liver) of sexually mature male and female M. chulae under solvent control (DMSO), bisphenol A (BPA), cadmium (Cd), and sulfamethazine (SMX) treatment conditions. Gene-expression stability was assessed using the comparative ΔCt method, NormFinder, geNorm, BestKeeper, and the integrated RefFinder ranking. The results showed that reference-gene stability was strongly tissue-specific and analysis-dependent. The integrated RefFinder analysis identified eif3h, rpl7, rps4x, stau1, and ef1y as the most stable genes in the pooled dataset, whereas ube2, hprt1l, and aldob were the least stable. geNorm analysis indicated that two reference genes were sufficient for brain, gill, intestine, and liver, whereas four were required for the gonad and six for cross-tissue comparisons. These findings provide the first systematic basis for reference-gene selection in M. chulae and establish an important methodological foundation for future qRT-PCR studies in this species, particularly in research on endocrine disruption, reproductive physiology, and marine ecotoxicology. Full article

Michelia compressa, a member of the Magnoliaceae family, is an evergreen tree of considerable significance in both landscape gardening and industrial production. However, during its introduction to northern subtropical regions in China, this species often suffers from frost damage, which limits its widespread application. The utilization of housekeeping genes is essential when performing gene family analyses under abiotic stress conditions. Additionally, auxin response factor (ARF) transcription factors (TF) play a crucial role in plant responses to abiotic stresses; however, their specific function in cold stress responses within M. compressa has not been systematically investigated. Ten housekeeping genes were selected from transcriptome data for evaluation using quantitative real-time PCR (qRT-PCR). The optimal housekeeping gene identified through screening was used for verification of gene family analysis. Additionally, key genes underwent functional validation. Analysis conducted with GeNorm, NormFinder, and BestKeeper identified 28S as the optimal reference gene for M. compressa under cold stress. Furthermore, an analysis of the ARF gene family using full-length transcriptome data revealed a total of 48 McoARF genes, which clustered into three groups alongside the Arabidopsis thaliana ARFs. Among these, eight selected McoARF genes exhibited significantly elevated expression levels in leaves under cold stress and demonstrated tissue specificity. Functional validation revealed that transgenic plants overexpressing McoARF13 displayed elevated levels of reactive oxygen species (ROS), hydrogen peroxide (H₂O₂), and malondialdehyde (MDA), as well as increased activities of peroxidase (POD) and superoxide dismutase (SOD) in leaves under cold stress. This study represents the inaugural screening of housekeeping genes in M. compressa under cold stress conditions, accompanied by an analysis of the ARF gene family. The functional validation of McoARF13 was successfully conducted, offering valuable insights into the molecular mechanisms that underlie cold stress response in M. compressa. Full article

Urinary microRNAs (miRNAs) are promising noninvasive biomarkers for cancer detection, but their clinical utility is reduced by inconsistent normalization strategies, reducing reproducibility and comparability across studies. In this study, we assessed the stability of miR-20a as an endogenous normalizer for urinary miRNA profiling in clear cell renal cell carcinoma (ccRCC) while standardizing the pre-analytical phase using a urine stabilizing solution. Ninety-nine urine samples were analyzed: 47 from healthy individuals, 30 from ccRCC patients pre-surgery, and 22 post-operative patients. Six candidate miRNAs—miR-20a, miR-15b, miR-16, miR-15a, miR-210-3p, and miR-let-7b—were quantified via RT-qPCR. Stability analysis with RefFinder, integrating multiple algorithms (geNorm, normFinder, BestKeeper, and ΔCt methods), identified miR-20a as the most stable among the six candidates. Raw Ct values of miR-20a were normally distributed (Shapiro–Wilk test, p > 0.05), with no significant intergroup differences (one-way ANOVA, F(2.96) = 2.324, p = 0.103) and minimal intragroup variability (CV% 4.98–6.38). MiR-20a expression remained stable across different tumor staging, grading, and urine storage durations. These findings confirm miR-20a as a robust endogenous normalizer for urinary miRNA analyses and support the feasibility of developing reproducible urinary liquid biopsy workflows for ccRCC, even in settings where immediate sample processing is not feasible. Full article

Backgrounds: Roegneria ciliaris is a perennial tetraploid wild relative of wheat that is widely distributed in China. It can be used both as a forage crop and ecological grass (the grasses specifically bred for ecological restoration) due to its strong stress tolerance, early green-up, vigorous seedling growth in spring, and great palatability. Methods: It is necessary to select and validate appropriate reference genes (RGs) for gene expression normalization by qRT-PCR in order to decipher the stress tolerance mechanism of this grass species. Therefore, eight candidate RGs were identified from transcriptome data of R. ciliaris ‘Liao sheng’ in response to drought stress. The expression stability of these RGs was evaluated by five algorithms (∆Ct, geNorm, NormFinder, Bestkeeper and ReFinder) using samples from different tissues and drought stress. Results: The results showed that MDH and RPL19 were the most stable RGs among all samples, while GAPDH and TUBA presented the lowest expression stability. These representative RGs were further used to normalize the expression level of the pyrroline-5-carboxylate synthase (P5CS) and protein phosphatase 2C (PP2C) genes in different tissues and under drought stress. The results of P5CS and PP2C expression were consistent with transcriptome data. Conclusion: Our study provided the first systematic evaluation of the most stable RG selection for qRT-PCR normalization in R. ciliaris, which will promote further research on its tissue-specific gene expression and mechanism of drought tolerance. Full article

Accurate normalization of RT-qPCR data requires selecting stable internal control genes, particularly in models characterized by dynamic metabolic transitions, such as 3T3-L1 adipocytes. The current study compares the expression stability of nine widely used housekeeping genes (HKGs) (peptidylprolyl isomerase A (Ppia), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), beta-2 microglobulin (B2M), ribosomal protein, large, P0 (36b4), hydroxymethylbilane synthase (Hmbs), hypoxanthine guanine phosphoribosyl transferase (Hprt), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (Ywhaz), 18S ribosomal RNA (18S), and β-actin (Actb)) across key stages of differentiation (days 0, 9, and 18) and under treatments with palmitic acid and docosahexaenoic acid. Stability was assessed using four classical algorithms—geNorm, NormFinder, BestKeeper, and RefFinder—supplemented by the ΔCt method, conventional statistical testing, correlation, and regression analysis relative to two target genes, fatty acid-binding protein 4 (Fabp4) and sterol regulatory element binding transcription factor 1 (Srebf1). The obtained data indicate that no single HKG remains universally stable across these experimental conditions, and the expression of traditionally used reference genes (Gapdh, Actb, Hprt, 18S) is highly influenced by both the stage of adipogenesis and exposure to lipid-modulating factors. In contrast, Ppia, 36b4, and B2M—despite some of them being underestimated in use as references—consistently display the lowest variability across most analytical tools, forming a reliable and functionally diverse normalization panel. It should be noted that our initial stability assessment revealed apparent discrepancies among mathematical evaluation methods, emphasizing the need for a holistic, multiple-level approach strategy. The applied combination of algorithmic and statistical methods provides a more rigorous and objective framework for assessing the stability of reference genes, which is highly recommended in such a complex adipocyte-based model. Full article

The cotton bollworm (Helicoverpa armigera, Lepidoptera: Noctuidae) is a globally distributed agricultural pest. When conducting expression analysis of its functional genes, appropriate reference genes should be selected to ensure the reliability of the results. In this study, five algorithms including Delta Ct, GeNorm, Normfinder, BestKeeper, and RefFinder were used to evaluate the expression stability of eleven candidate reference genes under different developmental stages, larval tissues, adult sexes, plant secondary substance stresses, and insecticide treatments in H. armigera. The candidate genes included Actin, Tubulin, EF-1α, RPS3, RPS15, RPL27, RPL32, 28S, GAPDH, SOD, and TRX. The reliability of the recommended reference gene combinations was validated using the growth arrest and DNA-damage-inducible gene 45 (GADD45). The results showed that normalizing relative expression of the target gene with the combination of the two most stable reference genes is recommended. Specifically, the combination of RPS3 + RPL27 is recommended for developmental stage comparisons; RPL32 + RPL27 for larval tissues; RPS3 + RPL27 for adult sex comparisons; GAPDH + RPL32 under tannic acid stress; RPL32 + RPS3 under quercetin stress; RPS15 + RPL32 under 2-tridecanone stress; RPS3 + RPL32 under ZQ-8 stress; RPL27 + TRX following chlorantraniliprole treatment; and RPL27 + RPL32 following indoxacarb treatment. Moreover, larvae exposed to three concentrations of plant secondary substances and to sublethal doses of insecticides exhibited significant upregulation of GADD45: after 4 h of exposure to 1% tannic acid, 0.1% and 1% quercetin, 1% 2-tridecanone, and 0.05% ZQ-8; after 15 h of chlorantraniliprole treatment; and after 24 h of indoxacarb treatment. Thus, GADD45 was overexpressed in response to various plant secondary substances and insecticide treatments, indicating its involvement in the detoxification and metabolism of H. armigera. This study proves to be helpful for selecting reference genes in H. armigera under plant secondary substance and insecticide stress and lays the foundation for further research utilizing GADD45 as a molecular target for pest control. Full article

Quantitative real-time PCR (qPCR) remains a cornerstone method for analyzing gene expression due to its high sensitivity, specificity, and reproducibility. However, for reliable results in relative quantification studies, the choice of an appropriate reference gene is critical to ensure accurate normalization. The expression of commonly used reference genes can vary depending on developmental stage and experimental conditions, making their validation essential. To date, no validated reference genes have been reported for Agrostemma githago L. (corn cockle, Caryophyllaceae). To facilitate research on genes involved in natural product biosynthesis and specialized metabolism regulation, we aimed to identify the most stable reference genes across various plant organs and cultivation conditions of this species. Drawing on previous literature, we have selected seven housekeeping genes widely used for evaluation: actin, β-tubulin, elongation factor 1α, glyceraldehyde-3-phosphate dehydrogenase, histone H3, translation elongation factor 1, and eukaryotic translation initiation factor 5A1 (for which two primer sets were tested). The nucleotide sequences of these potential reference genes were identified from the A. githago transcriptome. Using qRT-PCR, transcript levels of seven potential reference genes were estimated in 40 different A. githago samples, including 25 in vitro samples under various treatment conditions and 15 soil-grown samples representing A. githago organs in different developmental stages. Expression stability of candidate reference genes was assessed using the RefFinder platform, which combines four commonly applied statistical algorithms: geNorm, NormFinder, BestKeeper, and the comparative Δ-Ct method. The results revealed that the selection of optimal reference genes varied based on the particular organ, developmental stage and condition being examined. TIF5A1-2 (one of the two primer pairs tested) and GAPHD consistently exhibited the most stable expression under various conditions in vitro. EF1α and H3 exhibited superior performance across different organs of soil-grown plants. Moreover, our integrated analysis enabled the identification of the two most stable, universal reference genes suitable for normalization in A. githago under all tested conditions—H3 and TIF5A1-2. Our work provides a robust foundation for future transcriptomic and functional studies of the specialized metabolism of A. githago and other related species. Full article

Background/Objectives: Advanced 3D cell culture techniques enhance the physiological relevance of in vitro models, while supporting the 3Rs principles (Reduction, Refinement, and Replacement) of animal experimentation. In this context, 3D collagen-based systems mimic key extracellular matrix properties, enabling more accurate cellular organization and phenotype. However, changes in culture dimensionality can affect RT-qPCR reference gene stability, underscoring the need for careful validation when combining 2D and 3D systems. Methods: AML12 cells were cultured for 7 days under different 2D and collagen-based 3D conditions. The expression stability of nine candidate housekeeping genes was systematically evaluated using established algorithms (BestKeeper, NormFinder, geNorm, RefFinder, and ΔCt method), followed by inter-group statistical and correlation analyses of raw Ct values. Albumin gene expression was used as a target gene. Results: Although all candidate genes initially met acceptable variability thresholds, a stepwise, exclusion-based analysis revealed distinct performance differences. Hprt, Ppia, and Actb emerged as the most stable, showing no intra-group variability or interaction with Albumin expression. Nevertheless, Ywhaz and Rplp0, despite their high stability, were compromised by significant correlation with Albumin. Furthermore, Ywhaz showed significant downregulation under 3D culture conditions. B2M, Gapdh, 18S, and Hmbs exhibited increased variability, likely reflecting metabolic and microenvironmental heterogeneity associated with prolonged 2D cultivation of AML12 cells. Conclusions: Overall, this study highlights the importance of context-dependent, exclusion-based reference gene validation when comparing 2D and 3D models, and demonstrates a new approach for reliable gene expression normalization in complex in vitro culture systems. Full article

Quantitative real-time PCR (qRT-PCR) is a high-reliability, -sensitivity, and -operability technique to quantify gene expression. It is necessary to select stable reference genes for normalization. Water plays important roles in the metabolism, physiology, distribution, and so on, in insects. In this study, the suitability of various reference genes for qRT-PCR analysis was evaluated in different developmental stages of Chilo suppressalis exposed to desiccation or rehydration stress. The ∆Ct method, geNorm, NormFinder, and BestKeeper were used to evaluate the suitability of nine reference genes for normalizing gene expression in the third instar larvae, the fifth instar larvae, male pupae, female pupae, male adults, and female adults under different humidities. The results indicated that 18S rRNA was the most stable reference gene for monitoring gene expression in the third instar larvae, while ACTIN, TUB, UBI, UBI, and EF1 were the optimal genes for the fifth instar larvae, male pupae, female pupae, male adults, and female adults, respectively. The optimal number of reference genes recommended by geNorm analysis indicated that two candidate reference genes were sufficient for data normalization under all experimental conditions tested. To validate these recommendations, the expression profile of the gene encoding heat shock protein 60 (Hsp60) was investigated. Hsp60 transcript levels showed significant differences when normalized to the most stable single reference gene, or combined reference genes, compared with the least stable reference gene. The reference genes identified in the present study will enhance the reliability of gene expression data for C. suppressalis under humidity stress. Full article

Background/Objectives: The selection and validation of species-specific housekeeping genes (HKGs) have become increasingly common in functional genomics, with application of quantitative Polymerase Chain Reaction (qPCR) or cDNA-based qPCR (RT-qPCR). Despite the Macrobrachium amazonicum having RNA-seq studies available, there are still no data on the most stable and consistent HKGs for use in relative gene expression analyses. Therefore, the present study aimed to identify and validate seven HKGs in M. amazonicum: Eukaryotic Translation Initiation Factor (EIF), 18S ribosomal RNA (18S), Ribosomal Protein L18 (RPL18), β-actin, α-tubulin (α-tub), Elongation Factor 1-α (EF-1α), and Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH). Methods: The HKGs were identified in the M. amazonicum transcriptome, characterized for identity confirmation, and compared against public databases. Subsequently, RT-qPCR assays were prepared using muscle, hepatopancreas, gills, testis, androgenic gland, and ovary to assess the stability of the HKG markers, employing the comparative ∆Ct, BestKeeper, NormFinder, and GeNorm methods. Results: All candidate HKGs identified showed high similarity with other decapods. Reactions performed with these markers demonstrated high specificity, PCR efficiency, and elevated coefficients of determination. The comprehensive ranking, indicated that no single HKG was stable across all tissues, with HKGs showing the best stability being tissue-specific. The most stable HKGs were RPL18 and 18S. GAPDH, historically used as an HKG, showed the poorest performance in stability ranking for most tissues tested, whereas β-actin was most suitable only for ovarian. Conclusions: These data reinforce the need for species-specific HKG validation and provide an appropriate panel of reference markers for gene expression studies in the M. amazonicum. Full article

Fig (Ficus carica L.), a deciduous fruit tree that belongs to the Moraceae family, is cultivated worldwide as an important fruit crop for raw and processed foods. Quantitative real-time PCR (RT-qPCR) is a widely used method in F. carica to elucidate expression of genes related to various physiological responses. However, no studies have identified appropriate reference genes for RT-qPCR normalization in F. carica. In this study, 12 genes were selected from the F. carica genome as candidate reference genes for normalizing target gene expression. All candidate genes exhibited high amplification efficiency and specificity in the absence of primer dimers or extra PCR amplicons. The expression levels of the candidate genes were measured in three different plant tissues (fruit, leaf, and stem) under fungal pathogen infection using RT-qPCR. Their expression stabilities were evaluated using four computational algorithms: geNorm, Normfinder, delta-Ct, and BestKeeper. The RefFinder program was also used to calculate the geometric mean of the stability rankings obtained from these algorithms. The comprehensive ranking revealed that FcYLS8, FcPP2A, and FcAP2M were the most stable reference genes under biotic stress in the fruits, leaves, and stems, respectively. In contrast, traditional reference genes such as FcACT2, FcEF-1α, FcGAPDH, FcUBC21, and FcUBQ5 exhibited relatively low expression stability in all tested tissues. This study identified and validated stable reference genes for RT-qPCR normalization in F. carica, thus providing a valuable resource for accurate gene expression studies under biotic stress and highlighting the importance of validating reference genes to ensure reliable and reproducible RT-qPCR analysis. Full article

In this study, we focused on the screening and identification of reference genes for Paracarophenax alternatus Xu and Zhang. The laboratory population was used as the laboratory population, and samples were collected from mites at four different stages, including physogastry, viviparous, 5 d viviparous and phoresy. Then, the expression levels of seven candidate reference genes (α-tubulin, β-tubulin, RPS18, RPL13, GAPDH, EF1A, SDHA) were detected through qRT-PCR. Melting curves showed good gene specificity, and the amplification efficiency ranged from 90% to 102%. ΔCt analysis indicated that GAPDH was the most stable reference gene. The GeNorm software determined that the optimal number of reference genes was two, with GAPDH and RPS18 forming the most stable combination, and NormFinder identified RPS18 as the most stable reference gene. Although the BestKeeper software suggested that EF1A was the most stable, its p-value exceeded 0.05, rendering it unsuitable for use as a reference gene. Finally, through the RefFinder network tool, the most stable reference genes were identified as GAPDH and RPS18. Full article