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The liver plays a vital role in lipid synthesis and metabolism in poultry. To study the functional genes more effectively, it is essential to screen of reliable reference genes in the chicken liver, including females, males, embryos, as well as the Leghorn Male Hepatoma (LMH) cell line. Traditional reference gene screening involves selecting commonly used housekeeping genes (HKGs) for RT-qPCR experiments and using different algorithms to identify the most stable ones. However, this approach is limited in selecting the best reference gene from a small pool of HKGs. High-throughput sequencing technology may offer a solution to this limitation. This study aimed to identify the most consistently expressed genes by utilizing multiple published RNA-seq data of chicken liver and LMH cells. Subsequently, the stability of the newly identified reference genes was assessed in comparison to previously validated stable poultry liver expressed reference genes and the commonly employed HKGs using RT-qPCR. The findings indicated that there is a higher degree of similarity in stable expression genes between female and male liver (such as LSM14A and CDC40). In embryonic liver, the optimal new reference genes were SUDS3, TRIM33, and ERAL1. For LMH cells, the optimal new reference genes were ALDH9A1, UGGT1, and C21H1orf174. However, it is noteworthy that most HKGs did not exhibit stable expression across multiple samples, indicating potential instability under diverse conditions. Furthermore, RT-qPCR experiments proved that the stable expression genes identified from RNA-seq data outperformed commonly used HKGs and certain validated reference genes specific to poultry liver. Over all, this study successfully identified new stable reference genes in chicken liver and LMH cells using RNA-seq data, offering researchers a wider range of reference gene options for RT-qPCR in diverse situations.
Subject(s)
Chickens , Genes, Essential , Liver , Real-Time Polymerase Chain Reaction , Reference Standards , Animals , Chickens/genetics , Liver/metabolism , Male , Female , Real-Time Polymerase Chain Reaction/standards , Real-Time Polymerase Chain Reaction/methods , Gene Expression Profiling/standards , Gene Expression Profiling/methods , Cell Line, Tumor , Chick EmbryoABSTRACT
Analysis of gene expression is a pivotal method at the core of biomarkers studies and cancer research. Currently, RT-qPCR is the most commonly used technique to investigate the expression of certain genes. The accurate and reliable result relies on an effective normalization step using suitable reference genes. The present study was designed to evaluate the eligibility of a set of candidate mRNAs and snoRNA as reference genes in the most common human thyroid neoplasms. We tested the expression levels of eleven mRNA and small RNA housekeeping genes in thyroid samples. The stability of the candidate genes was examined in different thyroid lesions and under different experimental conditions. Results were compared to the reported data in the TCGA database. Our results suggested HPRT1 and ACTB as the best mRNA reference genes, SNORD96A, and SNORD95 as the best miRNA reference genes in thyroid tissues. These genes showed the most stable expression pattern among different thyroid lesions as well as different experimental conditions. The findings in this study highlight the effect of reference genes selection on data interpretation and emphasize the importance of testing for suitable reference genes to be used in specific types of cells and experimental conditions to ensure the validity and accuracy of results.
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BACKGROUND: A correct and stably expressing reference gene is prerequisite for successful quantitative real-time PCR (qRT-PCR). Investigating gene expression profiling during flower development could enhance our understanding of the molecular mechanisms of flower formation and fertility in Lycium. METHODS AND RESULTS: In this study, 11 candidate reference genes in Lycium flower development were selected from transcriptome sequence data and evaluated with five traditional housekeeping genes from previous studies based on qRT-PCR amplification. Comparing the expression stability result of 16 candidate genes using GeNorm, NormFinder, BestKeeper, and Delta Ct algorithms, Lba04g01649 and Lba12g02820 were validated as the optimal reference genes for the flower development of Lycium. CONCLUSIONS: The reference genes identified in this study would improve the accuracy of qRT-PCR quantification of target gene expression in Lycium flower development and facilitate future functional genomics studies on flower development. This research could lay the foundation for the study of the reproduction and development of the Lycium flower.
Subject(s)
Flowers , Gene Expression Profiling , Gene Expression Regulation, Plant , Genes, Plant , Lycium , Real-Time Polymerase Chain Reaction , Reference Standards , Lycium/genetics , Lycium/growth & development , Flowers/genetics , Flowers/growth & development , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Gene Expression Regulation, Plant/genetics , Gene Expression Profiling/methods , Gene Expression Profiling/standards , Transcriptome/genetics , Genes, Essential/genetics , Hybridization, GeneticABSTRACT
Chilo sacchariphagus (Lepidoptera: Pyralidae) is an economically important sugarcane pest. Although numerous studies were conducted on the physiological responses in C. sacchariphagus, little is known regarding the genes regulating these physiological processes. Gene expression analysis by qRT-PCR can offer a significant indication for functional gene studies. To our knowledge, the reference genes of C. sacchariphagus have not been screened or evaluated, which hinders the functional gene study. In the present study, the stability of seven reference genes (ß-ACT, GAPDH, BTF3, 28S, RPL7, EF1α, and SDHA) was evaluated in C. sacchariphagus under different experimental conditions, including tissues (antenna, head, thorax, abdomen, leg, and wing), temperatures (4 °C, 25 °C, and 37 °C) and sexes (male and female), through RefFinder, which integrates four algorithms (Normfinder, BestKeeper, ΔCt method, and geNorm). The findings suggested that the combination of ß-ACT and RPL7 is ideal to analyze gene expressions in different tissues and at distinct temperatures, and EF1α and SDHA were suitable reference genes for comparing gene expressions between sexes. Finally, the expression profiles of CsacPBP1 gene were evaluated, and the outcomes further confirm the importance of selecting fitting reference genes for normalization of qRT-PCR data. This study represents the first kind in screening out suitable reference genes for gene expression analysis in C. sacchariphagus. Information from this study is poised to galvanize future inquiry into the gene expression of C. sacchariphagus, an economically important pest of sugarcane.
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Сoding sequences of seven housekeeping genes: actin SaACT7, ubiquitin-conjugating protein SaUBC10, glyceraldehyde-3-phosphate dehydrogenase SaGAPDH, protein of the large subunit of ribosomes SaL2, α-tubulin SaTUA, translation elongation factor SaeEF1α, and protein phosphatase SaPP2A were identified as candidate reference genes for expression analysis of target genes in the extremely salt tolerant plant Suaeda altissima (L.) Pall. The expression profiles of the genes differed. SaACT7 and SaeEF1α demonstrated the highest expression levels, while the lowest expression levels were found for SaPP2A and SaTUA. SaPP2A and SaeEF1α genes were the most stably expressed at different steady-state salinity levels and different nitrate concentrations in nutrient solutions (NSs). SaL2, SaPP2A, and SaeEF1α genes showed the greatest stability of expression when nitrate was added to nutrient solution of plants grown under conditions of nitrate deficiency. Less constant expression was demonstrated in this experiment by SaACT7 and SaTUA. SaL2, SaACT7, SaeEF1α, and SaUBC10 genes showed the smallest expression changes under salt shock. To validate the use of the most stably expressed genes for normalization of gene expression, we checked them as reference genes to study the expression of the nitrate transporter gene SaNPF6.3 in S. altissima roots under conditions of different salinity and different nitrate supply.
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BACKGROUND: The robustness and credibility of RT-qPCR results are critically dependent on the selection of suitable reference genes. However, the mineralization of the extracellular matrix can alter the intracellular tension and energy metabolism within cells, potentially impacting the expression of traditional reference genes, namely Actb and Gapdh. OBJECTIVE: To methodically identify appropriate reference genes for research focused on mouse cementoblast mineralization. MATERIALS AND METHODS: Time-series transcriptomic data of mouse cementoblast mineralization were used. To ensure expression stability and medium to high expression levels, three specific criteria were applied to select potential reference genes. The expression stability of these genes was ranked based on the DI index (1/coefficient of variation) to identify the top six potential reference genes. RT-qPCR validation was performed on these top six candidates, comparing their performance against six previously used reference genes (Rpl22, Ppib, Gusb, Rplp0, Actb, and Gapdh). Cq values of these 12 genes were analyzed by RefFinder to get a stability ranking. RESULTS: A total of 4418 (12.27%) genes met the selection criteria. Among them, Rab5if, Chmp4b, Birc5, Pea15a, Nudc, Supt4a were identified as candidate reference genes. RefFinder analyses revealed that two candidates (Birc5 and Nudc) exhibited superior performance compared to previously used reference genes. LIMITATIONS: RefFinder's stability ranking does not consider the influence of primer efficiency. CONCLUSIONS AND IMPLICATIONS: We propose Birc5 and Nudc as candidate reference genes for RT-qPCR studies investigating mouse cementoblast mineralization and cementum repair.
Subject(s)
Dental Cementum , Real-Time Polymerase Chain Reaction , Survivin , Animals , Mice , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Survivin/genetics , Reference Standards , RNA-Seq/methods , RNA-Seq/standards , Calcification, Physiologic/geneticsABSTRACT
BACKGROUND: Real-time quantitative PCR (RT-qPCR) is one of the most widely used gene expression analyses for validating RNA-seq data. This technique requires reference genes that are stable and highly expressed, at least across the different biological conditions present in the transcriptome. Reference and variable candidate gene selection is often neglected, leading to misinterpretation of the results. RESULTS: We developed a software named "Gene Selector for Validation" (GSV), which identifies the best reference and variable candidate genes for validation within a quantitative transcriptome. This tool also filters the candidate genes concerning the RT-qPCR assay detection limit. GSV was compared with other software using synthetic datasets and performed better, removing stable low-expression genes from the reference candidate list and creating the variable-expression validation list. GSV software was used on a real case, an Aedes aegypti transcriptome. The top GSV reference candidate genes were selected for RT-qPCR analysis, confirming that eiF1A and eiF3j were the most stable genes tested. The tool also confirmed that traditional mosquito reference genes were less stable in the analyzed samples, highlighting the possibility of inappropriate choices. A meta-transcriptome dataset with more than ninety thousand genes was also processed successfully. CONCLUSION: The GSV tool is a time and cost-effective tool that can be used to select reference and validation candidate genes from the biological conditions present in transcriptomic data.
Subject(s)
Real-Time Polymerase Chain Reaction , Reference Standards , Software , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Animals , RNA-Seq/methods , RNA-Seq/standards , Gene Expression Profiling/methods , TranscriptomeABSTRACT
Urinary extracellular vesicles (uEVs) are rich in valuable biomolecule information which are increasingly recognized as potential biomarkers for various diseases. uEV long RNAs are among the critical cargos capable of providing unique transcriptome information of the source cells. However, consensus regarding ideal reference genes for relative long RNAs quantification in uEVs is not available as of date. Here we explored stable reference genes through profiling the long RNA expression by RNA-seq following unsupervised analysis and validation studies. Candidate reference genes were identified using four algorithms: NormFinder, GeNorm, BestKeeper and the Delta Ct method, followed by validation. RNA profile showed uEVs contained abundant long RNAs information and the core transcriptome was related to cellular structures, especially ribosome which functions mainly as translation, protein and RNA binding molecules. Analysis of RNA-seq data identified RPL18A, RPL11, RPL27, RACK1, RPSA, RPL41, H1-2, RPL4, GAPDH, RPS27A as candidate reference genes. RT-qPCR validation revealed that RPL41, RPSA and RPL18A were reliable reference genes for long RNA quantification in uEVs from patients with diabetes mellitus (DM), diabetic nephropathy (DN), IgA nephropathy (IgAN) and prostate cancer (PCA). Interestingly, RPL41 also outperformed traditional reference genes in renal tissues of DN and IgAN, as well as in plasma EVs of several types of cancers. The stable reference genes identified in this study may facilitate development of uEVs as novel biomarkers and increase the accuracy and comparability of biomarker studies.
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The present study was conducted to understand transcriptional response of skin fibroblast of yak (Bos grunniens) and cows of Bos indicus origin to hypoxia stress. Six primary fibroblast cell lines derived from three individuals each of Ladakhi yak (Bos grunniens) and Sahiwal cows (Bos indicus) were exposed to low oxygen concentration for a period of 24 h, 48 h and 72 h. The expression of 10 important genes known to regulate hypoxia response such as HIF1A, VEGFA, EPAS1, ATP1A1, GLUT1, HMOX1, ECE1, TNF-A, GPx and SOD were evaluated in fibroblast cells of Ladakhi yak (LAY-Fb) and Sahiwal cows (SAC-Fb) during pre- and post-hypoxia stress. A panel of 10 reference genes (GAPDH, RPL4, EEF1A1, RPS9, HPRT1, UXT, RPS23, B2M, RPS15, ACTB) were also evaluated for their expression stability to perform accurate normalization. The expression of HIF1A was significantly (p < 0.05) induced in both LAY-Fb (2.29-fold) and SAC-Fb (2.07-fold) after 24 h of hypoxia stress. The angiogenic (VEGFA), metabolic (GLUT1) and antioxidant genes (SOD and GPx) were also induced after 24 h of hypoxia stress. However, EPAS1 and ATP1A1 induced significantly (p < 0.05) after 48 h whereas, ECE1 expression induced significantly (p < 0.05) at 72 h after exposure to hypoxia. The TNF-alpha which is a pro-inflammatory gene induced significantly (p < 0.05) at 24 h in SAC-Fb and at 72 h in LAY-Fb. The induction of hypoxia associated genes indicated the utility of skin derived fibroblast as cellular model to evaluate transcriptome signatures post hypoxia stress in populations adapted to diverse altitudes.
Subject(s)
Altitude , Fibroblasts , Hypoxia , Skin , Animals , Cattle , Fibroblasts/metabolism , Skin/metabolism , Hypoxia/genetics , Tropical Climate , Female , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Stress, Physiological/geneticsABSTRACT
The Enterobacter cancerogenus strain EcHa1 was isolated from the dead larvae of Helicoverpa armigera, and has the potential for biocontrol of some Lepidoptera insects. In order to screen insecticidal-related genes by qRT-PCR, stable endogenous reference genes used for normalizing qRT-PCR data were selected and evaluated from 13 housekeeping genes (HKGs). The expression levels of the HKGs were determined using qRT-PCR under different experimental conditions, including two culture temperatures and three bacterial OD values. Five stability analysis methods (Ct, BestKeeper, NormFinder, geNorm, and RefFinder) were used to comprehensively rank the candidate genes. The results showed that the optimal reference genes varied under different experimental conditions. The combination of gyrA and gyrB was recommended as the best reference gene combination at 28 °C, while gyrA and rpoB was the best combination at 37 °C. When the OD values were 0.5, 1.0 and 2.0, the recommended reference gene combinations were ftsZ and gyrA, rpoB and gyrB, and gyrA and pyk, respectively. The most suitable reference genes were gyrA and gyrB under all experimental conditions. Using gyrA and gyrB as the reference genes for qRT-PCR, EcHa1 was found to invade all tissues of the H. armigera larvae, and expressed a candidate pathogenic factor Hcp at high levels in gut, Malpighian tubules, and epidermis tissues. This study not only establishes an accurate and reliable normalization for qRT-PCR in entomopathogenic bacteria but also lays a solid foundation for further study of functional genes in E. cancerogenus.
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Phaseolus vulgaris L., commonly known as the common bean, is a highly nutritious crop often called the "poor man's meat". However, it is susceptible to various diseases throughout the cropping season, with anthracnose caused by Colletotrichum lindemuthianum being a significant threat that leads to substantial losses. There is still a lack of understanding about the molecular basis of C. lindemuthianum pathogenicity. The first step in understanding this is to identify pathogenicity genes that express more during infection of common beans. A reverse transcription quantitative real-time PCR (qPCR) method can be used for virulence gene expression. However, this approach requires selecting appropriate reference genes to normalize relative gene expression data. Currently, there is no reference gene available for C. lindemuthianum. In this study, we selected eight candidate reference genes from the available genome of C. lindemuthianum to bridge the gap. These genes were ACT (Actin), ß-tub (ß-tubulin), EF (Elongation Factor), Cyt C (Cytochrome C), His H3 (Histone H3), CHS1 (Chitin synthetase), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) and abfA (Alpha-l-Arabinofuranosidase A). The primers for these candidate reference genes were able to amplify cDNA only from the pathogen, demonstrating their specificity. The qPCR efficiency of the primers ranged from 80% to 103%. We analyzed the stability of gene expression in C. lindemuthianum by exposing the mycelium to nine different stress conditions. We employed algorithms, such as GeNorm, NormFinder, BestKeeper, and RefFinder tools, to identify the most stable gene. The analysis using these tools revealed that EF, GAPDH, and ß-tub most stable genes, while ACT and CHS1 showed relatively low expression stability. A large number of potential effector genes have been identified through bioinformatics analysis in C. lindemuthianum. The stable genes for qPCR (EF and GAPDH) discovered in this study will aid the scientific community in determining the relative expression of C. lindemuthianum effector genes.
Subject(s)
Colletotrichum , Phaseolus , Plant Diseases , Real-Time Polymerase Chain Reaction , Reference Standards , Colletotrichum/genetics , Phaseolus/microbiology , Real-Time Polymerase Chain Reaction/methods , Real-Time Polymerase Chain Reaction/standards , Plant Diseases/microbiology , Gene Expression Profiling , Genes, FungalABSTRACT
Trichomonas gallinae, a globally distributed protozoan parasite, significantly affects the pigeon-breeding industry. T. gallinae infection mainly causes yellow ulcerative nodules on the upper respiratory tract and crop mucosa of pigeons, impeding normal breathing and feeding and ultimately causing death. Real-time quantitative PCR (qPCR) is a crucial technique for gene-expression analysis in molecular biology. Reference-gene selection for normalization is critical for ensuring this technique's accuracy. However, no systematic screening or validation of T. gallinae reference genes has been reported. This study quantified the transcript levels of ten candidate reference genes in T. gallinae isolates with different genotypes and culture conditions using qPCR. Using the geNorm, NormFinder, and BestKeeper algorithms, we assessed these reference genes' stabilities and ranked them using RankAggreg analysis. The most stable reference gene was tubulin beta chain (TUBB), while the widely used reference genes TUBG and GAPDH demonstrated poor stability. Additionally, we evaluated these candidate reference genes' stabilities using the T. gallinae TgaAtg8 gene. On using TUBB as a reference gene, TgaAtg8's expression profiles in T. gallinae isolates with different genotypes remained relatively consistent under various culture conditions. Conversely, using ACTB as a reference gene distorted the data. These findings provide valuable reference-gene-selection guidance for functional gene research and gene-expression analysis in T. gallinae.
Subject(s)
Columbidae , Reference Standards , Stress, Physiological , Trichomonas , Trichomonas/genetics , Animals , Columbidae/genetics , Columbidae/parasitology , Stress, Physiological/genetics , Gene Expression Profiling/methods , Real-Time Polymerase Chain Reaction/standards , Real-Time Polymerase Chain Reaction/methods , Tubulin/genetics , Trichomonas Infections/parasitology , Trichomonas Infections/veterinary , Genes, Protozoan , GenotypeABSTRACT
Recently, an increasing number of studies have investigated the mechanism of action of lactobacilli in the treatment of non-alcoholic fatty liver disease. Using four computational tools (NormFinder, geNorm, Delta Ct, and BestKeeper), six potential reference genes (RGs) were analyzed in the human liver cell line HepG2 cultivated 24 h in the presence of two strains of heat-killed lactobacilli, Limosilactobacillus reuteri E and Lactiplantibacillus plantarum KG4, respectively, in different cultivation media [Dulbecco´s Modified Eagle´s Medium (DMEM) high glucose or Roswell Park Memorial Institute (RPMI)]. The analysis revealed that the suitability of RG was similar between the two lactobacilli but quite different between the two media. The commonly used RGs, 18S rRNA and glyceraldehyde-3-phosphate dehydrogenase were the most unstable in DMEM high glucose. Normalization of the mRNA expression of the target gene encoding sterol regulatory element-binding protein 1c (SREBP-1c) to different RGs resulted in different expression profiles. This demonstrates that validation of candidate RGs under specific experimental conditions is crucial for the correct interpretation of quantitative polymerase chain reaction data. In addition, the choice of media has a profound impact on the effect of lactobacilli on lipogenesis at the gene expression level, as shown by the transcription factor SREBP-1c.
Subject(s)
Culture Media , Humans , Culture Media/chemistry , Hep G2 Cells , Lactobacillus/genetics , Real-Time Polymerase Chain Reaction , Sterol Regulatory Element Binding Protein 1/genetics , Sterol Regulatory Element Binding Protein 1/metabolism , Reference Standards , Gene Expression ProfilingABSTRACT
Circulating cell-free microRNAs (miRNAs) are promising biomarkers for medical decision-making. Suitable endogenous controls are essential to ensure reproducibility. We aimed to identify and validate endogenous reference miRNAs for qPCR data normalization in samples from SARS-CoV-2-infected hospitalized patients. We used plasma samples (nâ¯=â¯170) from COVID-19 patients collected at hospital admission (COVID-Ponent project, www.clinicaltrials.gov/NCT04824677). First, 179 miRNAs were profiled using RT-qPCR. After stability assessment, candidates were validated using the same methodology. miRNA stability was analyzed using the geNorm, NormFinder and BestKeeper algorithms. Stability was further evaluated using an RNA-seq dataset derived from COVID-19 hospitalized patients, along with plasma samples from patients with critical COVID-19 profiled using RT-qPCR. In the screening phase, after strict control of expression levels, stability assessment selected eleven candidates (miR-17-5p, miR-20a-5p, miR-30e-5p, miR-106a-5p, miR-151a-5p, miR-185-5p, miR-191-5p, miR-423-3p, miR-425-5p, miR-484 and miR-625-5p). In the validation phase, all algorithms identified miR-106a-5p and miR-484 as top endogenous controls. No association was observed between these miRNAs and clinical or sociodemographic characteristics. Both miRNAs were stably detected and showed low variability in the additional analyses. In conclusion, a 2-miRNA panel composed of miR-106a-5p and miR-484 constitutes a first-line normalizer for miRNA-based biomarker development using qPCR in hospitalized patients infected with SARS-CoV-2.
Subject(s)
Biomarkers , COVID-19 , MicroRNAs , SARS-CoV-2 , Humans , COVID-19/genetics , COVID-19/diagnosis , Biomarkers/blood , SARS-CoV-2/genetics , MicroRNAs/blood , MicroRNAs/genetics , Male , Female , Middle Aged , Severity of Illness Index , Aged , Circulating MicroRNA/blood , Circulating MicroRNA/genetics , Adult , Reproducibility of ResultsABSTRACT
Diorhabda rybakowi Weise is one of the dominant pests feeding on Nitraria spp., a pioneer plant used for windbreaking and sand fixation purposes, and poses a threat to local livestock and ecosystems. To clarify the key olfactory genes of D. rybakowi and provide a theoretical basis for attractant and repellent development, the optimal reference genes under two different conditions (tissue and sex) were identified, and the bioinformatics and characterization of the tissue expression profiles of two categories of soluble olfactory proteins (OBPs and CSPs) were investigated. The results showed that the best reference genes were RPL13a and RPS18 for comparison among tissues, and RPL19 and RPS18 for comparison between sexes. Strong expressions of DrybOBP3, DrybOBP6, DrybOBP7, DrybOBP10, DrybOBP11, DrybCSP2, and DrybCSP5 were found in antennae, the most important olfactory organ for D. rybakowi. These findings not only provide a basis for further in-depth research on the olfactory molecular mechanisms of host-specialized pests but also provide a theoretical basis for the future development of new chemical attractants or repellents using volatiles to control D. rybakowi.
ABSTRACT
Real-time quantitative PCR (RT-qPCR) is a widely used method for accurate quantitative gene expression analysis. For accurate quantitative verification of RT-qPCR, it is essential to select a reference gene with high expression stability depending on the experimental environment or the different tissues. In this study, we evaluated the stability of nine candidate reference genes, labeled elongation factor 1-alpha (EF1A), ERBB receptor feedback inhibitor 1-like isoform x2 (ERRFI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), integrin beta2 like (ITGB2), phosphatidylinositol-binding clathrin assembly protein-like isoform x3 (PICALM), 60 s ribosomal protein L5 (RPL5), 60 s ribosomal protein L7 (RPL7), tubulin beta chain (TUBB), and ubiquitin-conjugating enzyme E2A (UBE2A), in the brain (including pituitary gland) gonads and caudal fins of silvertip tetra (Hasemania nana) males and females. The stability evaluation of the reference gene was analyzed using a program based on the geNorm, NormFinder, BestKeeper, and RankAggreg algorithms. As a result, RPL5 (brain, caudal fin), EF1A (gonad), and PICALM (three tissue types) genes were evaluated as the most stable genes in silvertip tetra females. In males, TUBB (brain, caudal fin) and ITGB2 (gonads, three tissue types) genes were the most stable, and in both sexes, TUBB (brain), ITGB2 (caudal fin), RPL5 (gonads), and PICALM (three tissue types) genes are considered appropriate as reference genes for qRT-PCR analysis. However, the GAPDH gene was judged to be inappropriate for use as a reference gene because gene stability in the brain, caudal fin, and gonads was evaluated to be low in all males and females. As an introductory study on silvertip tetra, a new research model fish, the results of this study are expected to provide helpful information regarding sex differentiation and determination in fish.
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Introduction: Bone, a pivotal structural organ, is susceptible to disorders with profound health implications. The investigation of gene expression in bone tissue is imperative, particularly within the context of metabolic diseases such as obesity and diabetes that augment the susceptibility to bone fractures. The objective of this study is to identify a set of internal control genes for the analysis of gene expression. Methods: This study employs reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) to assess gene expression in bone tissue. We selected fourteen housekeeping genes and assessed their stability in the cortical bone of mouse models for obesity and diabetes using four well-established algorithms (GeNorm, BestKeeper, NormFinder, and the comparative Delta Ct method). Results and Conclusion: We identified Rpl13a as the mostly stably expressed reference gene in cortical bone tissue from mouse models of obesity and diabetes (db/db), while Gapdh was found to be the most stable reference gene in another diabetes model, KKAy mice. Additionally, Ef1a, Ppia, Rplp0, and Rpl22 were identified as alternative genes suitable for normalizing gene expression in cortical bone from obesity and diabetes mouse models. These findings enhance RT-qPCR accuracy and reliability, offering a strategic guide to select reference gene for studying bone tissue gene expression in metabolic disorders.
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Quantitative real-time PCR (qRT-PCR) is a widely applied technique for accurately assessing the expression of target genes. In practice, the evaluation of gene expression requires appropriate reference genes. To screen reliable reference genes for evaluating gene expression via qRT-PCR in Mythimna loreyi, a notorious migratory pest across Asia, Africa, Europe, and Australia, we assessed the expression stability of 13 candidate reference genes in M. loreyi using the ΔCt method, BestKeeper, Normfinder, GeNorm, and the web-based comprehensive platform RefFinder. These reference genes include RPL10, RPL27, RPL32, RPS3, TATA-box, GAPDH, AK, Actin, EF, α-tubulin, SOD, 18S rRNA, and FTZ-F1, which is frequently employed in Lepidoptera insects. Our findings revealed that the performance of the candidate reference gene depended on experimental conditions. Specifically, RPL27 and RPL10 were the most suitable for evaluating expression changes across developmental stages, tissues, and adult ages. The optimal reference genes were recommended in specific experiment conditions, for instance, EF and RPS3 were recommended for mating status, AK and RPL10 were recommended for temperature treatments, RPL27 and FTZ-F1 were recommended for larva diet, and EF and RPL27 were recommended for adult diet treatments. Additionally, expression profiles of pheromone-binding protein 2 (MlorPBP2) and glutathione S-transferase (MlorGST1) were used to validate the reference genes. This study provides reference genes for the accurate normalization of qRT-PCR data, laying the groundwork for studying the expression of target genes in M. loreyi.
ABSTRACT
Real-time quantitative PCR (qRT-PCR) is a pivotal technique for gene expression analysis. To ensure reliable and accurate results, the internal reference genes must exhibit stable expression across varied experimental conditions. Currently, no internal reference genes for Camellia impressinervis have been established. This study aimed to identify stable internal reference genes from eight candidates derived from different developmental stages of C. impressinervis flowers. We employed geNorm, NormFinder, and BestKeeper to evaluate the expression stability of these candidates, which was followed by a comprehensive stability analysis. The results indicated that CiTUB, a tubulin gene, exhibited the most stable expression among the eight reference gene candidates in the petals. Subsequently, CiTUB was utilized as an internal reference for the qRT-PCR analysis of six genes implicated in the petal pigment synthesis pathway of C. impressinervis. The qRT-PCR results were corroborated by transcriptome sequencing data, affirming the stability and suitability of CiTUB as a reference gene. This study marks the first identification of stable internal reference genes within the entire genome of C. impressinervis, establishing a foundation for future gene expression and functional studies. Identifying such stable reference genes is crucial for advancing molecular research on C. impressinervis.
Subject(s)
Camellia , Camellia/genetics , Gene Expression Profiling/methods , Transcriptome , Real-Time Polymerase Chain Reaction/methods , Flowers/genetics , Reference StandardsABSTRACT
Extracellular vesicles (EVs) contain a plethora of biomolecules, including nucleic acids, with diverse diagnostic and therapeutic application potential. Although reverse transcription-quantitative PCR (RT-qPCR) is the most widely applied laboratory technique to evaluate gene expression, its applicability in EV research is challenged by the lack of universal and stably present reference genes (RGs). In this study, we identify, validate and establish SNRPG, OST4, TOMM7 and NOP10 as RGs for the normalization of EV-associated genes by RT-qPCR. We show the stable presence of SNRPG, OST4, TOMM7 and NOP10 in multiple cell lines and their secreted EVs (n = 12) under different (patho)physiological conditions as well as in human-derived biofluids (n = 3). Enzymatic treatments confirm the presence of SNRPG, OST4, TOMM7 and NOP10 inside EVs. In addition, the four EV-associated RGs are stably detected in a size-range of EV subpopulations. RefFinder analysis reveals that SNRPG, OST4, TOMM7 and NOP10 are more stable compared to RGs established specifically for cultured cells or tissues such as HMBS, YWHAZ, SDHA and GAPDH. In summary, we present four universal and stably present EV-associated RGs to enable normalization and thus steer the implementation of RT-qPCR for the analysis of EV-associated RNA cargo for research or clinical applications.