Reference genes for proximal femoral epiphysiolysis expression studies in broilers cartilage.
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ABSTRACT:
SUBMITTER: Hul LM
PROVIDER: S-EPMC7447007 | biostudies-literature | 2020
REPOSITORIES: biostudies-literature
Publications
Reference genes for proximal femoral epiphysiolysis expression studies in broilers cartilage.
Hul Ludmila Mudri LM Ibelli Adriana Mércia Guaratini AMG Peixoto Jane de Oliveira JO Souza Mayla Regina MR Savoldi Igor Ricardo IR Marcelino Débora Ester Petry DEP Tremea Mateus M Ledur Mônica Corrêa MC
PloS one 20200825 8
The use of reference genes is required for relative quantification in gene expression analysis and the stability of these genes can be variable depending on the experimental design. Therefore, it is indispensable to test the reliability of endogenous genes previously to their use. This study evaluated nine candidate reference genes to select the most stable genes to be used as reference in gene expression studies with the femoral cartilage of normal and epiphysiolysis-affected broilers. The femu ...[more]
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Project description:(1) Background: Since the large-scale poultry industry has been established, femoral head necrosis (FHN) has always been a major leg disease in fast-growing broilers worldwide. Previous research suggested that cartilage homeostasis could be taken into consideration in the cause of FHN, but the evidence is insufficient. (2) Methods: One-day-old broiler chickens were randomly divided into three groups, 16 broilers per group. The birds in group L were injected intramuscularly with methylprednisolone (MP) twice a week for four weeks (12.5 mg·kg-1). The birds in group H were injected intramuscularly with MP (20 mg·kg-1·d-1) for 7 d (impulse treatment). The birds in group C were treated with sterile saline as a control group. Broilers were sacrificed at 42 and 56 d. Blood samples were collected from the jugular vein for ELISA and biochemical analysis. Bone samples, including femur, tibia, and humerus, were collected for histopathological analysis, bone parameters detection, and real-time quantitative PCR detection. (3) Results: The FHN broilers in group L and H both showed lower body weight (BW) and reduced bone parameters. In addition, the MP treatment resulted in reduced extracellular matrix (ECM) anabolism and enhanced ECM catabolism. Meanwhile, the autophagy and apoptosis of chondrocytes were enhanced, which led to the destruction of cartilage homeostasis. Moreover, the impulse MP injection increased the portion of birds with severer FHN, whereas the MP injection over a long period caused a more evident change in serum cytokine concentrations and bone metabolism indicators. (4) Conclusions: The imbalance of cartilage homeostasis may play a critical role in the development of FHN in broilers. FHN broilers induced by MP showed a more pronounced production of catabolic factors and suppressed the anabolic factors, which might activate the genes of the WNT signal pathway and hypoxia-inducible factors (HIFs), and then upregulate the transcription expression of ECM to restore homeostasis.
| S-EPMC7402315 | biostudies-literature
Project description:To be accurate, quantitative Polymerase Chain Reaction (qPCR) studies require a set of stable reference genes for normalization. This is especially critical in cardiac research because of the diversity of the clinical and experimental conditions in the field. We analyzed the stability of previously described as potential reference genes in different subsets of cardiac tissues, each representing a different field in cardiac research. The qPCR dataset was based on 119 different tissue samples derived from cardiac development to pathology in mouse adult hearts. These samples were grouped into 47 tissue types. The stability of 9 candidate genes was analyzed in each of 12 experimental conditions comprising different groupings of these tissue types. Expression stability was determined with the geNorm module of qbase+. This analysis showed that different sets of two or three reference genes are required for analysis of qPCR data in different experimental conditions in murine cardiac research.
| S-EPMC5428317 | biostudies-literature
Project description:ObjectiveIdentification of genes differentially expressed in mechano-biological pathways in articular cartilage provides insight into the molecular mechanisms behind initiation and/or progression of osteoarthritis (OA). Quantitative PCR (qPCR) is commonly used to measure gene expression, and is reliant on the use of reference genes for normalisation. Appropriate validation of reference gene stability is imperative for accurate data analysis and interpretation. This study determined in vitro reference gene stability in articular cartilage explants and primary chondrocytes subjected to different compressive loads and tensile strain, respectively.DesignThe expression of eight commonly used reference genes (18s, ACTB, GAPDH, HPRT1, PPIA, RPL4, SDHA and YWHAZ) was determined by qPCR and data compared using four software packages (comparative delta-Ct method, geNorm, NormFinder and BestKeeper). Calculation of geometric means of the ranked weightings was carried out using RefFinder.ResultsAppropriate reference gene(s) for normalisation of mechanically-regulated transcript levels in articular cartilage tissue or isolated chondrocytes were dependent on experimental set-up. SDHA, YWHAZ and RPL4 were the most stable genes whilst glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to a lesser extent Hypoxanthine-guanine phosphoribosyltransferase (HPRT), showed variable expression in response to load, demonstrating their unsuitability in such in vitro studies. The effect of using unstable reference genes to normalise the expression of aggrecan (ACAN) and matrix metalloproteinase 3 (MMP3) resulted in inaccurate quantification of these mechano-sensitive genes and erroneous interpretation/conclusions.ConclusionThis study demonstrates that commonly used 'reference genes' may be unsuitable for in vitro cartilage chondrocyte mechanobiology studies, reinforcing the principle that careful validation of reference genes is essential prior to each experiment to obtain robust and reproducible qPCR data for analysis/interpretation.
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Project description:The choice of reference genes that are stably expressed amongst treatment groups is a crucial step in real-time quantitative PCR gene expression studies. Recent guidelines have specified that a minimum of two validated reference genes should be used for normalisation. However, a quantitative review of the literature showed that the average number of reference genes used across all studies was 1.2. Thus, the vast majority of studies continue to use a single gene, with β-actin (ACTB) and/or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) being commonly selected in studies of vertebrate gene expression. Few studies (15%) tested a panel of potential reference genes for stability of expression before using them to normalise data. Amongst studies specifically testing reference gene stability, few found ACTB or GAPDH to be optimal, whereby these genes were significantly less likely to be chosen when larger panels of potential reference genes were screened. Fewer reference genes were tested for stability in non-model organisms, presumably owing to a dearth of available primers in less well characterised species. Furthermore, the experimental conditions under which real-time quantitative PCR analyses were conducted had a large influence on the choice of reference genes, whereby different studies of rat brain tissue showed different reference genes to be the most stable. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies.
| S-EPMC4640531 | biostudies-literature
Project description:Quantitative real-time PCR (qPCR) rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian species other than the chicken has been investigated molecularly, highlighting the need for an experimentally validated pan-avian primer set for reference genes. Here we report testing a set for 14 candidate reference genes (18S, ABL, GAPDH, GUSB, HMBS, HPRT, PGK1, RPL13, RPL19, RPS7, SDHA, TFRC, VIM, YWHAZ) on different tissues of the mallard (Anas platyrhynchos), domestic chicken (Gallus gallus domesticus), common crane (Grus grus), white-tailed eagle (Haliaeetus albicilla), domestic turkey (Meleagris gallopavo f. domestica), cockatiel (Nymphicus hollandicus), Humboldt penguin (Sphenicus humboldti), ostrich (Struthio camelus) and zebra finch (Taeniopygia guttata), spanning a broad range of the phylogenetic tree of birds. Primer pairs for six to 11 genes were successfully established for each of the nine species. As a proof of principle, we analyzed expression levels of 10 candidate reference genes as well as FOXP2 and the immediate early genes, EGR1 and CFOS, known to be rapidly induced by singing in the avian basal ganglia. We extracted RNA from microbiopsies of the striatal song nucleus Area X of adult male zebra finches after they had sang or remained silent. Using three different statistical algorithms, we identified five genes (18S, PGK1, RPS7, TFRC, YWHAZ) that were stably expressed within each group and also between the singing and silent conditions, establishing them as suitable reference genes. In conclusion, the newly developed pan-avian primer set allows accurate normalization and quantification of gene expression levels in multiple avian species.
| S-EPMC4057121 | biostudies-literature
Project description:Necrosis of the femoral head (NFH), a severe orthopedic disease in adults, involves the collapse of the femoral head. The pathophysiological mechanisms underlying NFH are yet to be fully investigated. The aim of the present study was to identify potentially important genes and signaling pathways involved in NFH and investigate their molecular mechanisms. Gene expression profiles of patients with NFH and healthy controls were compared using the Gene Expression Omnibus (GEO) database repository of the National Center of Biotechnology Information. GSE74089 from the GEO database included 4 patients with NFH and 4 healthy individuals. A total of 1,191 differentially expressed genes (DEGs) were identified between the patients with NFH and controls, including 743 upregulated and 448 downregulated DEGs. Then, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that upregulated DEGs were mainly involved in the phosphoinositide 3‑kinase/protein kinase B signaling pathway, focal adhesion and extracellular matrix‑receptor interactions. Additionally, protein‑protein interaction (PPI) analysis identified the most central DEGs as vascular endothelial growth factor A, Jun proto‑oncogene, cyclin D1, fibroblast growth factor 2, HECT domain and ankyrin repeat‑containing E3 ubiquitin protein ligase 1, protein kinase Cα, bone morphogenetic protein 2 and prostaglandin‑endoperoxide synthase 2. PPI analysis also identified guanine nucleotide‑binding protein, γ13 as the most commonly downregulated gene based on different centrality. The results of the present study may provide novel insight into the genes and associated pathways involved in NFH, and aid the identification of novel therapeutic targets and biomarkers in the treatment of NFH.
| S-EPMC6691263 | biostudies-literature
Project description:BackgroundReverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) remains one of the best-established techniques to assess gene expression patterns. However, appropriate reference gene(s) selection remains a critical and challenging subject in which inappropriate reference gene selction can distort results leading to false interpretations. To date, mixed opinions still exist in how to choose the most optimal reference gene sets in accodrance to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guideline. Therefore, the purpose of this study was to investigate which schemes were the most feasible for the identification of reference genes in a bone and cartilage bioengineering experimental setting. In this study, rat bone mesenchymal stem cells (rBMSCs), skeletal muscle tissue and adipose tissue were utilized, undergoing either chondrogenic or osteogenic induction, to investigate the optimal reference gene set identification scheme that would subsequently ensure stable and accurate interpretation of gene expression in bone and cartilage bioengineering.ResultsThe stability and pairwise variance of eight candidate reference genes were analyzed using geNorm. The V0.15- vs. Vmin-based normalization scheme in rBMSCs had no significant effect on the eventual normalization of target genes. In terms of the muscle tissue, the results of the correlation of NF values between the V0.15 and Vmin schemes and the variance of target genes expression levels generated by these two schemes showed that different schemes do indeed have a significant effect on the eventual normalization of target genes. Three selection schemes were adopted in terms of the adipose tissue, including the three optimal reference genes (Opt3), V0.20 and Vmin schemes, and the analysis of NF values with eventual normalization of target genes showed that the different selection schemes also have a significant effect on the eventual normalization of target genes.ConclusionsBased on these results, the proposed cut-off value of Vn/n + 1 under 0.15, according to the geNorm algorithm, should be considered with caution. For cell only experiments, at least rBMSCs, a Vn/n + 1 under 0.15 is sufficient in RT-qPCR studies. However, when using certain tissue types such as skeletal muscle and adipose tissue the minimum Vn/n + 1 should be used instead as this provides a far superior mode of generating accurate gene expression results. We thus recommended that when the stability and variation of a candidate reference genes in a specific study is unclear the minimum Vn/n + 1 should always be used as this ensures the best and most accurate gene expression value is achieved during RT-qPCR assays.
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Project description:BACKGROUND: Sleep is a restorative process and is essential for maintenance of mental and physical health. In an attempt to understand the complexity of sleep, multidisciplinary strategies, including genetic approaches, have been applied to sleep research. Although quantitative real time PCR has been used in previous sleep-related gene expression studies, proper validation of reference genes is currently lacking. Thus, we examined the effect of total or paradoxical sleep deprivation (TSD or PSD) on the expression stability of the following frequently used reference genes in brain and blood: beta-actin (b-actin), beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine guanine phosphoribosyl transferase (HPRT). RESULTS: Neither TSD nor PSD affected the expression stability of all tested genes in both tissues indicating that b-actin, B2M, GAPDH and HPRT are appropriate reference genes for the sleep-related gene expression studies. In order to further verify these results, the relative expression of brain derived neurotrophic factor (BDNF) and glycerol-3-phosphate dehydrogenase1 (GPD1) was evaluated in brain and blood, respectively. The normalization with each of four reference genes produced similar pattern of expression in control and sleep deprived rats, but subtle differences in the magnitude of expression fold change were observed which might affect the statistical significance. CONCLUSION: This study demonstrated that sleep deprivation does not alter the expression stability of commonly used reference genes in brain and blood. Nonetheless, the use of multiple reference genes in quantitative RT-PCR is required for the accurate results.
| S-EPMC2685383 | biostudies-literature
Project description:Gene expression studies which utilize lipopolysaccharide (LPS)-stimulated macrophages to model immune signaling are widely used for elucidating the mechanisms of inflammation-related disease. When expression levels of target genes are quantified using Real-Time quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), they are analyzed in comparison to reference genes, which should have stable expression. Judicious selection of reference genes is, therefore, critical to interpretation of qRT-PCR results. Ideal reference genes must be identified for each experimental system and demonstrated to remain constant under the experimental conditions. In this study, we evaluated the stability of eight common reference genes: Beta-2-microglobulin (B2M), Cyclophilin A/Peptidylprolyl isomerase A, glyceraldehyde-3-phosphatedehydrogenase (GAPDH), Hypoxanthine Phosphoribosyltransferase 1, Large Ribosomal Protein P0, TATA box binding protein, Ubiquitin C (UBC), and Ribosomal protein L13A. Expression stability of each gene was tested under different conditions of LPS stimulation and compared to untreated controls. Reference gene stabilities were analyzed using Ct value comparison, NormFinder, and geNorm. We found that UBC, closely followed by B2M, is the most stable gene, while the commonly used reference gene GAPDH is the least stable. Thus, for improved accuracy in evaluating gene expression levels, we propose the use of UBC to normalize PCR data from LPS-stimulated macrophages.
| S-EPMC6994071 | biostudies-literature
Project description:BackgroundRelatively few studies have used cell culture systems to investigate gene expression and the regulation of myogenesis in fish. To produce robust data from quantitative real-time PCR mRNA levels need to be normalised using internal reference genes which have stable expression across all experimental samples. We have investigated the expression of eight candidate genes to identify suitable reference genes for use in primary myogenic cell cultures from Atlantic salmon (Salmo salar L.). The software analysis packages geNorm, Normfinder and Best keeper were used to rank genes according to their stability across 42 samples during the course of myogenic differentiation.ResultsInitial results showed several of the candidate genes exhibited stable expression throughout myogenic culture while Sdha was identified as the least stable gene. Further analysis with geNorm, Normfinder and Bestkeeper identified Ef1alpha, Hprt1, Ppia and RNApolII as stably expressed. Comparison of data normalised with the geometric average obtained from combinations of any three of these genes showed no significant differences, indicating that any combination of these genes is valid.ConclusionThe geometric average of any three of Hprt1, Ef1alpha, Ppia and RNApolII is suitable for normalisation of gene expression data in primary myogenic cultures from Atlantic salmon.
| S-EPMC2736950 | biostudies-literature