Search Results (164)

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

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

Reverse transcription quantitative PCR (RT-qPCR) is extensively used to quantify gene expression under drought conditions; however, its reliability depends on the validation of the reference genes under specific conditions. In cucumber, reference genes have rarely been validated under drought conditions. This study identified stable housekeeping genes for RT-qPCR normalization in the leaves of two inbred lines with contrasting drought responses. Plants underwent a 7-day drought period, with leaf samples collected at multiple points along with watered controls. The expression stability of 13 candidate genes was evaluated using four algorithms: geNorm, NormFinder, BestKeeper, and the comparative ΔCt method, with the results integrated using RefFinder. Ten genes producing specific and efficient amplicons were analyzed for stability. CACS and UBI-1 consistently ranked among the most stable genes, with TIP41-like as an additional reliable option, whereas GAPDH and HEL were unstable. GeNorm pairwise variation analysis showed that the two reference genes were sufficient for accurate normalization. Functional validation with three drought-responsive targets (LOX, HsfC1, and CYP72A219) and comparison with RNA sequencing (RNA-seq) fold changes confirmed that normalization using CACS and UBI-1 yielded the most biologically credible expression profiles. These reference genes will facilitate robust RT-qPCR analyses of drought response in cucumber leaves and provide a starting point for validating suitable normalizers in other cucumber organs and related cucurbits. Full article

Reverse transcription quantitative PCR (RT-qPCR) is a powerful and widely used technique for quantifying alterations in gene expression. Cassava bacterial blight caused by Xanthomonas phaseoli pv. manihotis severely constraints cassava growth and yield. Accurate evaluation of the expression levels of genes following infection by X. phaseoli pv. manihotis is crucial for the identification of potential cassava resistance genes. In this study, thirty-two novel potential reference genes were screened from the cassava–X. phaseoli pv. manihotis transcriptome. Their expression, along with that of seven literature-reported cassava reference genes, was evaluated in two susceptible and two resistant cassava varieties at six time points post-inoculation by X. phaseoli pv. manihotis through RT-qPCR analysis. The stability of thirty-nine candidate reference genes was assessed by four algorithms: geNorm, NormFinder, Delta Ct, and RefFinder. The results demonstrated that serving as new reference genes, MehnRNPR and MePRPF38B consistently exhibited superior expression stability over seven established reference genes under X. phaseoli pv. manihotis infection, regardless of the susceptible or resistant cassava varieties. The reliability of the reference genes was validated by assessing the expression pattern of MeNAC35 and MeSWEET10a under X. phaseoli pv. manihotis infection. The findings of this study provide valuable insights for advancing the precision of the quantification of cassava candidate genes associated with disease resistance. Full article

Accurate normalization of qRT-PCR data in pulmonary tuberculosis (TB) research requires reference genes whose expression is invariant across clinically relevant matrices, yet no studies have addressed this in lesion tissue and blood concurrently. We assessed the expression stability of eight popular housekeeping genes—ACTB, B2M, GAPDH, HPRT1, PPIA, RPL13A, UBC and YWHAZ—in lung tuberculomas and peripheral blood mononuclear cells (PBMCs) from TB patients. Standardized extraction and amplification yielded Cq values that were ranked by geNorm, NormFinder, BestKeeper and comparative Delta CT, with consensus scores generated in RefFinder; and correlation analysis was conducted in order to select the most suitable genes to work collectively for future normalization. The consensus analysis placed PPIA, YWHAZ and HPRT1 at the top, while GAPDH and UBC were the least stable. Our findings endorse a three-gene panel (PPIA, YWHAZ, HPRT1) for robust normalization of host gene-expression studies in both lesion tissue and PBMCs in pulmonary TB and highlight the necessity of context-specific reference-gene validation. Full article

Internal reference genes are a prerequisite for ensuring the accuracy of gene verification experiments, but few relevant studies on Lyophyllum decastes have investigated the growth cycle and different environmental conditions. In this study, the qPCR results of 22 house-keeping genes were analyzed using GeNorm, BestKeeper, NormFinder and RefFinder. The results revealed that the most stable gene differed under different conditions. Across all developmental stages and under hot, cold, acidic, alkaline, and salt conditions, UBCE gene displays the greatest expression stability. However, EF1b, β-ACT, HSD17B3, and Cyb presented the greatest stability under cold, heat, and acidic conditions, and heavy metal exposure, respectively. To screen for genes suitable for all conditions, RefFinder’s ranking results revealed that UBCE and EF1b ranked in the top 2, demonstrating the highest gene expression stability. In contrast, Cyb was positioned at the bottom of the comprehensive ranking table. This study not only revealed potential factors affecting the suitability of reference genes but also identified optimal reference genes from a set of candidate genes across diverse conditions. Full article

To obtain stable Chinese olive reference genes, eight genes (RPN2B, PIP1.4, NIFS1, RPS16, At5g12110, HSC-2, ABCG44, LOS1) exhibiting stable expression were identified as candidate reference genes from the transcriptome. The expression stability of these genes was evaluated across 33 Chinese olive fruit samples from different varieties and seven developmental stages. The most stable reference genes were determined through comparisons using ΔCq, geNorm, NormFinder, BestKeeper, and RefFinder. Analysis revealed that RPN2B and NIFS1 were consistently ranked among the most stable genes across the different algorithms and exhibited stable expression. Therefore, they are recommended as suitable reference genes for gene expression studies in Chinese olive fruits across different varieties and developmental stages. The four different methods of reference gene stability analysis were used to identify the most stable reference genes in different varieties and developmental stages of Chinese olive fruits, which can be used as a reference for the selection of reference genes in the subsequent gene expression studies of Chinese olive fruits. Full article

Indocalamus tessellatus (Munro) P. C. Keng is a bamboo species with significant economic and ecological value, and exhibits considerable resistance to abiotic stresses. However, systematic evaluation of reference genes for gene expression analysis in this species is lacking. Analysis of multi-tissue transcriptomes yielded 3801 relatively stable genes; from these, we selected eleven new candidates along with nine widely adopted reference genes. We then evaluated these candidates under four conditions: drought (15% PEG-6000), salt (200 mM NaCl), waterlogging (root submergence in water), and a multi-tissue panel (leaf, leaf sheath, culm, shoot, and root). Under stress, early and sustained time points were sampled to capture dynamic transcriptional responses. Expression stability was assessed using geNorm, NormFinder, BestKeeper, and ΔCt, and results were integrated with RefFinder to generate comprehensive stability rankings for each condition. The most stable reference genes were condition-dependent: MD10B and PP2A under drought, eIF1A and Ite23725 under salt stress, PP2A and eIF4A under waterlogging, and 60S and UBP1 across different tissues. Notably, commonly used genes such as UBI and Actin7 were less stable. Peroxidase (POD) was used as a validation marker because it is a known stress-responsive gene, providing a sensitive readout of normalization accuracy. Validation confirmed that selecting suitable reference genes is essential for dependable expression quantification. These findings provide a robust set of reference genes for qRT-PCR studies in I. tessellatus, supporting future molecular and functional research in bamboo. Full article

Salt-tolerance improvement of tomatoes is largely a task of modern selection and plant molecular genetics because of cultivation on dry and irrigated lands under salt stress. To reveal the salt resistance gene, we need quantitative real-time polymerase chain reaction (qRT-PCR) normalization through reference genes analysis. Sometimes, housekeeping gene expression changes in response to various stress factors, especially salinity. In this manuscript, we evaluated expression changes of elongation factor 1α X53043.1 (EF1α), actin BT013707.1 (ACT), ubiquitin NM_001346406.1 (UBI), nuclear transcript factor XM_026030313.2 (NFT-Y), β-tubulin NM_001247878.2 (TUB), glyceraldehyde-3 phosphate dehydrogenase NM_001247874.2 (GAPDH), phosphatase 2A catalytic subunit NM_001247587.2 (PP2a), and phosphoglycerate kinase XM_004243920.4 (PGK) in salt-sensitive Solanum lycopersicum L. YaLF line and salt tolerance Rekordsmen cv. under 100 mM NaCl. We also suggested potential correlations between relative water content (RWC), ion accumulation, and reference gene expression in tomato genotypes with contrasting salinity tolerance. We used geNorm, NormFinder, BestKeeper, ∆Ct, and RefFinder algorithms to establish a set of the most reliable tomato candidate genes. The most stable genes for YaLF tomatoes were ACT, UBI, TUB, and PP2a. Despite differences in ranks, the NFT-Y was present in Rekordsmen’s stable set. Full article

Salix suchowensis is an ideal model organism for investigating nitrogen (N) transport mechanisms due to its low N-input requirements. Accurate quantification of gene expression is essential for elucidating these processes, with quantitative real-time PCR (RT-qPCR) being the preferred method. However, the identification of stable reference genes for normalization in Salix suchowensis under varying N conditions remains unresolved. In this study, thirteen commonly employed candidate reference genes were evaluated across root, stem, and leaf tissues, under four N treatments (NH₄NO₃, NH₄⁺, NO₃⁻, and N deficiency). Five genes (UBQ1, UBQ3, 18S, H2A2, and H2B2) were excluded due to poor amplification efficiency or irregular melting curves. The remaining eight genes were further assessed for expression stability using the geNorm, NormFinder, and BestKeeper algorithms. Integrated ranking via RefFinder identified EF1α, EFβ, and αTUB as the most stable reference genes. GeNorm analysis suggested that two reference genes were sufficient for reliable normalization. Validation using the N-responsive gene SsAMT1 and SsNRT2 confirmed the stability of EF1α, EFβ, and αTUB as suitable reference genes. Based on comprehensive stability assessments and experimental validation, we recommended EF1α + αTUB as optimal reference gene pairs for RT-qPCR normalization under varying N conditions. Furthermore, the consistent expression of EF1α and αTUB across nine willow genotypes highlighted their broader applicability within Salix species. This study provides valuable methodological guidance for advancing molecular research on N transport in woody perennial plants. Full article

The present study aimed to identify genes encoding enzymes involved in the biotransformation of monoterpenoid (–)-isopulegol by Rhodococcus rhodochrous IEGM 1362. This strain is able to transform (–)-isopulegol with formation of two novel metabolites with promising antitumor and analeptic activities. Cell fractions of rhodococci and specific inhibitor of cytochrome P450-dependent oxygenase activity were used to establish the localization and type of biotransformation enzymes. The expression of nine CYP450 genes selected by bioinformatics analysis was analyzed by quantitative real-time PCR (qRT-PCR). Selection of optimal reference genes for normalization of qRT-PCR results was performed using BestKeeper, Normfinder, geNorm, Delta CT, and RefFinder algorithms. As a result of these studies, the role of CYP450 enzyme complexes in the biotransformation of (–)-isopulegol was confirmed, and their cytoplasmic localization was established. The genes encoding DNA gyrase subunit B (gyrB) and protein translocase subunit A (secA) were selected as the most stable reference genes. The induced expression of the gene encoding CYP450 hydroxylase in the presence of (–)-isopulegol was determined. The obtained data allow us to identify the specific CYP450 enzyme involved in (–)-isopulegol biotransformation by R. rhodochrous IEGM 1362 and lay the foundation for further studies of molecular and genetic mechanisms of monoterpenoid biotransformation. Full article