26S: Novel reference gene from leaves and roots of common bean for biotic stress expression expression studies based on PCR

The aim of this study was to evaluate the variation in the expression levels of five traditional housekeeping genes under contrasting conditions of abiotic stress to identify the most stable reference gene. In the evaluation of expression levels by RT-PCR, the gene with less variation among cultivars and treatments was the ribosomal 26S. Regarding to the variation between foliage and root tissues the more stable genes were â-tubulin and 26S, while between phenological stages the best were 26S and the 1-â elongation factor. The evaluation of expression levels by absolute qPCR confirmed that the 26S gene is a stable reference for the study of gene expression based on the PCR technique due to its low coefficient of variation through contrasting abiotic stress conditions.

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Identification of suitable reference genes of Scylla paramamosain for gene expression profiling in various tissues and under vibrio challenge

Crustaceana ◽

10.1163/15685403-00003823 ◽

2018 ◽

Vol 91 (10) ◽

pp. 1195-1210 ◽

Cited By ~ 2

Author(s):

Yabo Fang ◽

Le Diao ◽

Fengying Zhang ◽

Lingbo Ma ◽

Mengdi Zhao ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

18S Rrna ◽

Elongation Factor ◽

Scylla Paramamosain ◽

Mud Crab ◽

Expression Levels ◽

Qrt Pcr ◽

Elongation Factor 1

Abstract The quantitative real-time transcription-polymerase chain reaction (qRT-PCR) is now used widely in studies about mRNA expression levels. The selection of one or more stable reference gene(s) used for data normalization is substantial. In this study, expression levels of eleven candidate reference genes (β-actin, 16S rRNA, 18S rRNA, 28S rRNA, α-I tubulin, GAPDH, ribosomal protein L13, elongation factor 1 α, elongation factor 2, arginine kinase and ubiquitin) were examined using the GenomeLab GeXP analysis system (Beckman Coulter). Gene expression data were analysed using two different statistical models: geNorm and NormFinder. (1) In six different tissues (hepatopancreas, haemocytes, heart, gill, muscle, and testis) from the mud crab, Scylla paramamosain, 18S rRNA and elongation factor 1 α were identified as the two best reference genes. (2) In the haemocytes after being challenged by Vibro parahaemolyticus, the result suggested that ubiquitin was the most stable gene after the treatment. 18S rRNA, elongation factor 1 α and ubiquitin are herein recommended as the best combination. These results provide useful options for reference gene selection under different experimental conditions in qRT-PCR studies in the mud crab.

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Reference gene selection for quantitative RT-PCR in Miscanthus sacchariflorus under abiotic stress conditions

Molecular Biology Reports ◽

10.1007/s11033-021-06902-z ◽

2022 ◽

Author(s):

Junqin Zong ◽

Jingbo Chen ◽

Ling Li ◽

Jianjian Li ◽

Dandan Li ◽

...

Keyword(s):

Abiotic Stress ◽

Reference Gene ◽

Gene Selection ◽

Stress Conditions ◽

Rt Pcr ◽

Reference Gene Selection ◽

Miscanthus Sacchariflorus ◽

Selection For

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Identification of stable reference genes for lipopolysaccharide-stimulated macrophage gene expression studies

Biology Methods and Protocols ◽

10.1093/biomethods/bpw005 ◽

2016 ◽

Vol 1 (1) ◽

Cited By ~ 1

Author(s):

Roshini Kalagara ◽

Weimin Gao ◽

Honor L. Glenn ◽

Colleen Ziegler ◽

Laura Belmont ◽

...

Keyword(s):

Gene Expression ◽

Ribosomal Protein ◽

Reference Gene ◽

Reference Genes ◽

Target Genes ◽

Stable Gene ◽

Expression Levels ◽

Qrt Pcr ◽

Expression Studies ◽

Gene Expression Studies

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.

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Transcription Elongation Factor A-Like 7, regulated by miR-758-3p inhibits the malignant process of melanoma through decreasing the expression levels of c-Myc and AKT1

10.21203/rs.3.rs-86576/v1 ◽

2020 ◽

Author(s):

Xilin Liu ◽

Xianji Song ◽

Hong Li

Keyword(s):

Cancer Progression ◽

Transcription Elongation ◽

Ectopic Expression ◽

Elongation Factor ◽

Expression Level ◽

Rt Pcr ◽

Expression Levels ◽

Transcription Elongation Factor ◽

Melanoma Progression ◽

Underlying Mechanisms

Abstract Background: The ectopic expression of transcription elongation factor A (SII)-like 7 (TCEAL7) has been observed in several kinds of cancers, but its role in melanoma is still unclear. This study was carried out to investigate TCEAL7 role in melanoma progression, and uncover the underlying mechanisms. Methods: TCEAL7 expression level in melanoma tissues and cells were determined by using RT-PCR and western blotting. CCK-8, transwell chambers, flow cytometry, starch assay and tumorigenesis assay were applied to detect cell growth, invasion, apoptosis, migration and tumorigenesis, respectively. Results: A low expression level of TCEAL7 was observed in melanoma tissues and cells, which associated with malignant process and poor prognosis. TCEAL7 negatively modulated AKT1, AKT2 and c-Myc expression and inhibited cancer progression via decreasing AKT1 and c-Myc expression. In addition, TCEAL7 was negatively modulated by miR-758-3p which promoted melanoma progression. Moreover, TCEAL7 overexpression abolished miR-758-3p-mediated melanoma progression. Conclusion: This study demonstrated that TCEAL7, regulated by miR-758-3p inhibited the malignant process of melanoma through decreasing the expression levels of c-Myc and AKT1.

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Identification of Suitable Reference Genes for Expression Studies in Pomegranate Under Different Biotic and Abiotic Stress Conditions

10.21203/rs.3.rs-259407/v1 ◽

2021 ◽

Author(s):

Puspha Doddaraju ◽

Pavan Kumar ◽

Mahesh S Dashyal ◽

Manjunath Girigowda

Keyword(s):

Gene Expression ◽

Abiotic Stress ◽

Reference Gene ◽

Reference Genes ◽

Bacterial Blight ◽

Candidate Reference Gene ◽

Target Gene ◽

Stress Conditions ◽

Biotic And Abiotic Stress ◽

Expression Studies

Abstract Pomegranate (Punica granatum) is an important economic fruit crop, facing many biotic and abiotic challenges during cultivation. Several research programs are in progress to understand both biotic and abiotic stress factors and mitigate these challenges using gene expression studies based on the qPCR approach. However, research publications are not available yet to select the standard reference gene for normalizing target gene expression values in pomegranate. The most suitable candidate reference gene is required to ensure precise and reliable results for qPCR analysis. In the current research, eight candidate reference genes' stability was evaluated under different stress conditions using different algorithms such as ∆Ct, geNorm, BestKeeper, NormFinder and RefFinder. The various algorithms revealed that EFA1 and 18S rRNA were common and most stable reference genes (RGs) under abiotic and wilt stress. Whereas comprehensive ranking by RefFinder showed GAPDH and CYPF were the most stable RGs under combined biotic (pooled samples of all biotic stress) and bacterial blight samples. The two most stable reference genes are adequate for normalizing target gene expression under wilt, nematode, bacterial blight and abiotic stress conditions using qPCR. The above data provide comprehensive details for the selection of a candidate reference gene in various stresses in pomegranate

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CD34+ Cell Selection Is Required to Accurately Measure HOXA9 Levels by Quantitative RT-PCR in Patients with Myelodysplastic Syndrome.

Blood ◽

10.1182/blood.v104.11.4733.4733 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4733-4733

Author(s):

Jason N. Berman ◽

Stefan Heinrichs ◽

Taylor M. Ortiz ◽

Steven M. Kornblau ◽

Donna S. Neuberg ◽

...

Keyword(s):

Cell Separation ◽

Hox Genes ◽

Housekeeping Genes ◽

Housekeeping Gene ◽

Lymphoid Cells ◽

Cell Selection ◽

Rt Pcr ◽

Hematopoietic Stem ◽

Expression Levels ◽

Cd34 Cells

Abstract In recent years, it has become increasingly clear that overexpression of specific major HOX genes is linked to transformation of the malignant hematopoietic stem cells underlying human acute myeloid leukemias (AML) and myelodysplastic syndromes (MDS). For example, several recent studies indicate that overexpression of HOXA9 is associated with a poor outcome in AML and MDS. As we initiated our own studies of HOXA9 expression in MDS, we were concerned about the validity of studying whole bone marrow (BM) cell RNA obtained at diagnosis, due to the diversity of cell types and low blast cell numbers in the BM that characterize MDS, and prior studies of G. Savageau and K. Humphries demonstrating that HOXA9 expression in normal BM cells is limited to the CD34+ fraction, which is enriched for hematopoietic stem and progenitor cells. Thus, we initiated this study to investigate whether valid HOXA9 expression levels can be determined from whole BM cell RNAs of MDS patients or whether CD34+ cell separation is required prior to analysis. BM aspirate samples were collected at diagnosis from 9 patients with MDS and subjected to Ficoll-Hypaque light density cell separation, followed by magnetic bead separation to remove CD3+/CD19+ mature lymphoid cells. The CD3−/CD19− fraction was further fractionated into CD34+ and CD34− populations. RNA was extracted by standard methods and two-step quantitative real-time RT-PCR was performed using SYBR Green to measure HOXA9 and control housekeeping gene expression levels. RNAs from peripheral blood CD34+ cells from 8 healthy donors served as controls. Relative HOXA9 expression levels were obtained by normalization to three housekeeping genes (GAPDH, YWHAZ, and RPL4) for each fraction. Our results conclusively show that HOXA9 levels of CD34+ cells from MDS patients are on average three-fold higher than those of CD34− cells from the same patient (p=0.004, Wilcoxon signed rank). Thus, as is the case for normal BM cells, HOXA9 expression in MDS is highest in the stem and progenitor cell fraction. Our results indicate that CD34+ cell selection must be carried out to accurately assess the expression of HOXA9 in the malignant clone of MDS patients. Unselected BM cell measurements include variable fractions of CD34− and CD34+ cells, which can have significant variability, even in samples obtained from the same patient, thus confounding attempts to accurately measure HOXA9 levels and determine the contribution of this oncogene to MDS. Moreover, we detected levels of HOXA9 expression in the CD34+ cells of MDS patients that were on average 4.3 times those of normal controls (p=0.004, Wilcoxon rank sum). Our studies indicate that in order to advance understanding of the molecular pathogenesis of MDS, the expression levels of specific HOX genes and their co-regulators will need to be carefully evaluated in purified CD34+ BM cells from patients with this disease.

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Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity

BioMed Research International ◽

10.1155/2015/363575 ◽

2015 ◽

Vol 2015 ◽

pp. 1-20 ◽

Cited By ~ 6

Author(s):

Cora S. Thiel ◽

Swantje Hauschild ◽

Svantje Tauber ◽

Katrin Paulsen ◽

Christiane Raig ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Molecular Mechanisms ◽

Expression Profiles ◽

Housekeeping Genes ◽

Altered Gravity ◽

Experimental Conditions ◽

Expression Levels ◽

Expression Studies ◽

Gene Expression Studies

Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes (“housekeeping genes”) are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

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Assessment of common housekeeping genes as reference for gene expression studies using RT-qPCR in mouse choroid plexus

Scientific Reports ◽

10.1038/s41598-021-82800-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kim Hoa Ho ◽

Annarita Patrizi

Keyword(s):

Gene Expression ◽

Choroid Plexus ◽

Sensory Deprivation ◽

Housekeeping Genes ◽

Housekeeping Gene ◽

Experimental Conditions ◽

Brain Repair ◽

Expression Studies ◽

Testing Algorithms ◽

Gene Expression Studies

AbstractChoroid plexus (ChP), a vascularized secretory epithelium located in all brain ventricles, plays critical roles in development, homeostasis and brain repair. Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular and useful technique for measuring gene expression changes and also widely used in ChP studies. However, the reliability of RT-qPCR data is strongly dependent on the choice of reference genes, which are supposed to be stable across all samples. In this study, we validated the expression of 12 well established housekeeping genes in ChP in 2 independent experimental paradigms by using popular stability testing algorithms: BestKeeper, DeltaCq, geNorm and NormFinder. Rer1 and Rpl13a were identified as the most stable genes throughout mouse ChP development, while Hprt1 and Rpl27 were the most stable genes across conditions in a mouse sensory deprivation experiment. In addition, Rpl13a, Rpl27 and Tbp were mutually among the top five most stable genes in both experiments. Normalisation of Ttr and Otx2 expression levels using different housekeeping gene combinations demonstrated the profound effect of reference gene choice on target gene expression. Our study emphasized the importance of validating and selecting stable housekeeping genes under specific experimental conditions.

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Genome-wide identification and expression analysis of Raffinose synthetase family in cotton

BMC Bioinformatics ◽

10.1186/s12859-021-04276-4 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Ruifeng Cui ◽

Xiaoge Wang ◽

Waqar Afzal Malik ◽

Xuke Lu ◽

Xiugui Chen ◽

...

Keyword(s):

Abiotic Stress ◽

Expression Patterns ◽

Regulatory Elements ◽

Plant Leaves ◽

Motif Analysis ◽

Expression Levels ◽

Genome Wide ◽

Salt And Drought Stress ◽

Key Genes ◽

Stress Gene

Abstract Background The Raffinose synthetase (RAFS) genes superfamily is critical for the synthesis of raffinose, which accumulates in plant leaves under abiotic stress. However, it remains unclear whether RAFS contributes to resistance to abiotic stress in plants, specifically in the Gossypium species. Results In this study, we identified 74 RAFS genes from G. hirsutum, G. barbadense, G. arboreum and G. raimondii by using a series of bioinformatic methods. Phylogenetic analysis showed that the RAFS gene family in the four Gossypium species could be divided into four major clades; the relatively uniform distribution of the gene number in each species ranged from 12 to 25 based on species ploidy, most likely resulting from an ancient whole-genome polyploidization. Gene motif analysis showed that the RAFS gene structure was relatively conservative. Promoter analysis for cis-regulatory elements showed that some RAFS genes might be regulated by gibberellins and abscisic acid, which might influence their expression levels. Moreover, we further examined the functions of RAFS under cold, heat, salt and drought stress conditions, based on the expression profile and co-expression network of RAFS genes in Gossypium species. Transcriptome analysis suggested that RAFS genes in clade III are highly expressed in organs such as seed, root, cotyledon, ovule and fiber, and under abiotic stress in particular, indicating the involvement of genes belonging to clade III in resistance to abiotic stress. Gene co-expressed network analysis showed that GhRFS2A-GhRFS6A, GhRFS6D, GhRFS7D and GhRFS8A-GhRFS11A were key genes, with high expression levels under salt, drought, cold and heat stress. Conclusion The findings may provide insights into the evolutionary relationships and expression patterns of RAFS genes in Gossypium species and a theoretical basis for the identification of stress resistance materials in cotton.

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