Reference Genes for Quantitative qPCR Analyses in Monocytes of Septic Patients

2020 ◽  
Author(s):  
Raquel Bragante Gritte ◽  
Talita Souza-Siqueira ◽  
Laureane Nunes Masi ◽  
Juliana de Freitas Germano ◽  
Gilson Masahiro Murata ◽  
...  
Keyword(s):  
Reference Genes ◽  
Critical Role ◽  
Phosphoglycerate Kinase ◽  
Stable Gene ◽  
Essential Components ◽  
Single Reference Gene ◽  
Monocytes And Macrophages ◽  
Life Threatening ◽  
Suitable Combination ◽  
Suitable Reference

Abstract Background: Monocytes and macrophages are essential components of the innate and adaptive immune responses and play a critical role in sepsis. Sepsis is a life-threatening organ dysfunction associated with an unregulated host response to infection. About 20 million people develop sepsis annually, and up to 50% die. There is a lack of studies regarding human monocytes and sepsis. This study aimed to determine the most stable internal gene (s) to investigate gene expression in monocytes/macrophages of septic patients.Methods: The expression stability of fifteen commonly used reference genes was analyzed by determining the comparative threshold cycle (Ct) values, using the BestKeeper, GeNorm, and NormFinder algorithms.Results: BestKeeper analysis revealed that the syntaxin 5 (STX5A) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) genes were highly stable. GeNorm pointed out STX5A and phosphoglycerate kinase 1 (PGK1) as the most suitable combination whereas through NormFinder glyceraldehyde 3- phosphate dehydrogenase (GAPDH) and 14-3-3 zeta/delta protein (YWHAZ) was the most stable combination. All programs analysis discarded the use of heterogeneous nuclear ribonucleoprotein A/B (HNRNPAB). GeNorm and NormFinder indicated actin-beta (ACTB) as the least stable gene.Conclusions: The combined data indicated that STX5A, PGK1, GAPDH, and HPRT1 are highly suitable reference genes for qPCR analysis of septic patients monocytes. In the case of choosing one single reference gene, the results point out to STX5A (first place by GeNorm and BestKeeper and third place by NormFinder). This study is the first report on reference genes in monocytes/macrophages from septic patients.

scholarly journals Reference Genes for Quantitative Real-time Polymerase Chain Reaction (qPCR)) Analyses in Freshly Isolated Monocytes of Septic Patients

2021 ◽  
Author(s):  
Raquel Bragante Gritte ◽  
Talita Souza-Siqueira ◽  
Laureane Nunes Masi ◽  
Juliana Freitas Germano ◽  
Gilson Masahiro Murata ◽  
...  
Keyword(s):  
Reference Genes ◽  
Phosphoglycerate Kinase ◽  
Gene Expressions ◽  
Monocytes And Macrophages ◽  
Life Threatening ◽  
Internal Gene ◽  
Suitable Combination ◽  
Suitable Reference ◽  
Nuclear Ribonucleoprotein

Abstract Background: Sepsis is a life-threatening organ dysfunction associated with unregulated host response to infection. About 20 million people develop sepsis annually, and up to 50% die. Monocytes and macrophages play a key role in the innate and adaptive immune responses but the fully role of these cells in patients with sepsis still remains to be investigated. One of the limitations for the studies of gene expression in monocytes/macrophages in sepsis is the choice of the reference genes. We determined herein the most stable internal gene (s) to investigate gene expressions in monocytes/macrophages of septic patients.Methods: The expression stability of fifteen commonly used reference genes was analyzed by determining the comparative threshold cycle (Ct) values, using the BestKeeper, GeNorm, and NormFinder algorithms.Results: BestKeeper analysis revealed that the syntaxin 5 (STX5A) and hypoxanthine phosphoribosyltransferase 1 (HPRT1) genes are highly stable. GeNorm pointed out STX5A and phosphoglycerate kinase 1 (PGK1) as the most suitable combination, whereas through NormFinder glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and 14-3-3 zeta/delta protein (YWHAZ) was the most stable combination. All program analyses discarded the use of heterogeneous nuclear ribonucleoprotein A/B (HNRNPAB). GeNorm and NormFinder indicated actin-beta (ACTB) as the minor stable gene.Conclusions: The combined data indicated that STX5A, PGK1, GAPDH, and HPRT1 are highly suitable reference genes for qPCR analysis of septic patient monocytes. In choosing one reference gene, the results point out STX5A (first place by GeNorm and BestKeeper and third place by NormFinder). This study is the first report on reference genes in freshly obtained monocytes/macrophages from septic patients.

scholarly journals Evaluation of potential reference genes for quantitative RT-PCR analysis in spotted sea bass (Lateolabrax maculatus) under normal and salinity stress conditions

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5631 ◽  
Author(s):  
Haolong Wang ◽  
Haishen Wen ◽  
Yun Li ◽  
Kaiqiang Zhang ◽  
Yang Liu
Keyword(s):  
Salinity Stress ◽  
Reference Genes ◽  
18S Rrna ◽  
Sea Bass ◽  
Stress Conditions ◽  
Pcr Analysis ◽  
Stable Gene ◽  
Qrt Pcr ◽  
Lateolabrax Maculatus ◽  
Suitable Reference

The aim of this study was to select the most suitable reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) of spotted sea bass (Lateolabrax maculatus), an important commercial marine fish in Pacific Asia, under normal physiological and salinity stress conditions. A total of 9 candidate reference genes (HPRT, GAPDH, EF1A, TUBA, RPL7, RNAPol II, B2M, ACTB and 18S rRNA) were analyzed by qRT-PCR in 10 tissues (intestine, muscle, stomach, brain, heart, liver, gill, kidney, pectoral fins and spleen) of L. maculatus. Four algorithms, geNorm, NormFinder, BestKeeper, and comparative ΔCt method, were used to evaluate the expression stability of the candidate reference genes. The results showed the 18S rRNA was most stable in different tissues under normal conditions. During salinity stress, RPL7 was the most stable gene according to overall ranking and the best combination of reference genes was RPL7 and RNAPol II. In contrast, GAPDH was the least stable gene which was not suitable as reference genes. The study showed that different algorithms might generate inconsistent results. Therefore, the combination of several reference genes should be selected to accurately calibrate system errors. The present study was the first to select reference genes of L. maculatus by qRT-PCR and provides a useful basis for selecting the appropriate reference gene in L. maculatus. The present study also has important implications for gene expression and functional genomics research in this species or other teleost species.

scholarly journals Quantitative Evaluation and Selection of Reference Genes for Quantitative RT-PCR in Mouse Acute Pancreatitis

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Zhaoping Yan ◽  
Jinhang Gao ◽  
Xiuhe Lv ◽  
Wenjuan Yang ◽  
Shilei Wen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Pancreatitis ◽  
Reference Gene ◽  
Reference Genes ◽  
Suitable Reference Gene ◽  
Expression Stability ◽  
Rt Pcr ◽  
Comprehensive Method ◽  
Suitable Combination ◽  
Suitable Reference

The analysis of differences in gene expression is dependent on normalization using reference genes. However, the expression of many of these reference genes, as evaluated by quantitative RT-PCR, is upregulated in acute pancreatitis, so they cannot be used as the standard for gene expression in this condition. For this reason, we sought to identify a stable reference gene, or a suitable combination, for expression analysis in acute pancreatitis. The expression stability of 10 reference genes (ACTB, GAPDH, 18sRNA, TUBB, B2M, HPRT1, UBC, YWHAZ, EF-1α, and RPL-13A) was analyzed using geNorm, NormFinder, and BestKeeper software and evaluated according to variations in the raw Ct values. These reference genes were evaluated using a comprehensive method, which ranked the expression stability of these genes as follows (from most stable to least stable): RPL-13A, YWHAZ > HPRT1 > GAPDH > UBC > EF-1α> 18sRNA > B2M > TUBB > ACTB. RPL-13A was the most suitable reference gene, and the combination of RPL-13A and YWHAZ was the most stable group of reference genes in our experiments. The expression levels of ACTB, TUBB, and B2M were found to be significantly upregulated during acute pancreatitis, whereas the expression level of 18sRNA was downregulated. Thus, we recommend the use of RPL-13A or a combination of RPL-13A and YWHAZ for normalization in qRT-PCR analyses of gene expression in mouse models of acute pancreatitis.

scholarly journals The crucial choice of reference genes: identification of miR-191-5p for normalization of miRNAs expression in bone marrow mesenchymal stromal cell and HS27a/HS5 cell lines

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
É. Costé ◽  
F. Rouleux-Bonnin
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Reference Genes ◽  
Critical Role ◽  
Optimal Number ◽  
Stable Gene ◽  
Mirnas Expression ◽  
Post Transcriptional Regulation ◽  
Normalization Process ◽  
Hematopoietic Niche

Abstract Bone marrow mesenchymal stromal cells (BM-MSCs) have a critical role in tissue regeneration and in the hematopoietic niche due to their differentiation and self-renewal capacities. These mechanisms are finely tuned partly by small non-coding microRNA implicated in post-transcriptional regulation. The easiest way to quantify them is RT-qPCR followed by normalization on validated reference genes (RGs). This study identified appropriate RG for normalization of miRNA expression in BM-MSCs and HS27a and HS5 cell lines in various conditions including normoxia, hypoxia, co-culture, as model for the hematopoietic niche and after induced differentiation as model for regenerative medicine. Six candidates, namely miR-16-5p, miR-34b-3p, miR-103a-3p, miR-191-5p, let-7a-5p and RNU6A were selected and their expression verified by RT-qPCR. Next, a ranking on stability of the RG candidates were performed with two algorithms geNorm and RefFinder and the optimal number of RGs needed to normalize was determined. Our results indicate miR-191-5p as the most stable miRNA in all conditions but also that RNU6a, usually used as RG is the less stable gene. This study demonstrates the interest of rigorously evaluating candidate miRNAs as reference genes and the importance of the normalization process to study the expression of miRNAs in BM-MSCs or derived cell lines.

Validation of reference genes for ovarian tissue from capuchin monkeys (Cebus apella)

Zygote ◽  
2012 ◽  
Vol 21 (2) ◽  
pp. 167-171 ◽  
Author(s):  
A.B. Brito ◽  
J.S. Lima ◽  
D.C. Brito ◽  
L.N. Santana ◽  
N.N. Costa ◽  
...  
Keyword(s):  
Reference Genes ◽  
Target Genes ◽  
Ovarian Tissue ◽  
Rna Extraction ◽  
Cebus Apella ◽  
Capuchin Monkeys ◽  
Stable Gene ◽  
Polymerase Chain ◽  
The Stability ◽  
Suitable Reference

SummaryThere is no tradition in studies reporting the effect of exposure to cryoprotectants or simply hypoxia and hypothermia on gene expression in the ovarian tissue and there has been only one study on reference or target genes quantification, and comparisons of normoxic with hypoxic, hypothermic and toxic conditions. Our aim in the present study was to investigate the stability of three reference genes in the ovarian tissue of capuchin monkeys (Cebus apella). To this end, fresh and cryoprotectant-exposed ovarian biopsies were used. Both fresh and exposed ovarian tissues were subjected to total RNA extraction and synthesis of cDNA. cDNA was amplified by real-time polymerase chain reaction (PCR), and GeNorm, BestKeeper and NormFinder software were used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and TATA-binding protein (TBP). Results demonstrated that, in the ovarian tissue from capuchin monkeys, HPRT1 and TBP were the most suitable reference genes and thus could be used as parameters to normalize data in future studies. In contrast, GAPDH appeared as the least stable gene among the tested reference genes. In conclusion, HPRT1 and TBP were the most stable reference genes in fresh and cryoprotectant-exposed ovarian tissue from capuchin monkeys.

Selection and validation of reference genes for RT-qPCR analysis in Litchi chinensis Sonn. pericarp

2020 ◽  
Author(s):  
Fang Li ◽  
Jinhua Sun ◽  
Jiali Men ◽  
Huanling Li ◽  
Guo Wang ◽  
...  
Keyword(s):  
Reference Gene ◽  
Reference Genes ◽  
Negative Impact ◽  
Pcr Analysis ◽  
Stable Gene ◽  
Litchi Chinensis ◽  
Qrt Pcr ◽  
Pericarp Browning ◽  
Suitable Reference ◽  
Litchi Pericarp

Abstract Background: Quantitative real time PCR (qRT-PCR) is an important tool for gene expression analysis and function identification. Suitable reference genes are the basis of accurate and reliable qPCR results. Litchi ( Litchi chinensis Sonn.) is a commercially important tropical and subtropical fruit crop, rapid pericarp browning is the major negative impact on the industry. Reference gene validation would help screen for genes involved in the browning mechanism.Results: In this study, fifteen new candidate reference genes, identified with transcriptome data, were assessed to determine stable reference genes for qRT-PCR analysis of litchi pericarps from different varieties, with differing postharvest storage, and under pathogenic inoculation. Ct values, Genorm, Normfind, and Reffinder algorithms, were used to identify the most stable genes. The results showed that GAGA-25 was the most stable gene for comparing different varieties of fresh pericarp, HDAC9 was the most stable gene for postharvest pericarp, STAM was the most stable for inoculated pericarp. Among the candidate reference genes, GAGA-25 was the most stable reference gene across the complete sample set.Conclusion: This study evaluated reference gene stability for qRT-PCR with litchi pericarp. This work supplies a foundation for qPCR use in future gene function and molecular mechanism studies of litchi pericarp browning.

scholarly journals Structure, Function, and Regulation of the Essential Virulence Factor Capsular Polysaccharide of Vibrio vulnificus

2020 ◽  
Vol 21 (9) ◽  
pp. 3259 ◽  
Author(s):  
Gregg S. Pettis ◽  
Aheli S. Mukerji
Keyword(s):  
Coastal Waters ◽  
Capsular Polysaccharide ◽  
Vibrio Vulnificus ◽  
Critical Role ◽  
Phase Variation ◽  
Innate Immune ◽  
Repeat Sequences ◽  
Life Threatening ◽  
Inflammatory Cytokine Response ◽  
Group 1

Vibrio vulnificus populates coastal waters around the world, where it exists freely or becomes concentrated in filter feeding mollusks. It also causes rapid and life-threatening sepsis and wound infections in humans. Of its many virulence factors, it is the V. vulnificus capsule, composed of capsular polysaccharide (CPS), that plays a critical role in evasion of the host innate immune system by conferring antiphagocytic ability and resistance to complement-mediated killing. CPS may also provoke a portion of the host inflammatory cytokine response to this bacterium. CPS production is biochemically and genetically diverse among strains of V. vulnificus, and the carbohydrate diversity of CPS is likely affected by horizontal gene transfer events that result in new combinations of biosynthetic genes. Phase variation between virulent encapsulated opaque colonial variants and attenuated translucent colonial variants, which have little or no CPS, is a common phenotype among strains of this species. One mechanism for generating acapsular variants likely involves homologous recombination between repeat sequences flanking the wzb phosphatase gene within the Group 1 CPS biosynthetic and transport operon. A considerable number of environmental, genetic, and regulatory factors have now been identified that affect CPS gene expression and CPS production in this pathogen.

scholarly journals Selection of reference genes for gene expression analysis in Liriodendron hybrids’ somatic embryogenesis and germinative tissues

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tingting Li ◽  
Weigao Yuan ◽  
Shuai Qiu ◽  
Jisen Shi
Keyword(s):  
Gene Expression ◽  
Somatic Embryogenesis ◽  
Reference Genes ◽  
Gene Selection ◽  
Elongation Factor ◽  
Reverse Transcription Pcr ◽  
Expression Of Genes ◽  
Suitable Reference ◽  
Functional Analyses ◽  
Elongation Factor 1

AbstractThe differential expression of genes is crucial for plant somatic embryogenesis (SE), and the accurate quantification of gene expression levels relies on choosing appropriate reference genes. To select the most suitable reference genes for SE studies, 10 commonly used reference genes were examined in synchronized somatic embryogenic and subsequent germinative cultures of Liriodendron hybrids by using quantitative real-time reverse transcription PCR. Four popular normalization algorithms: geNorm, NormFinder, Bestkeeper and Delta-Ct were used to select and validate the suitable reference genes. The results showed that elongation factor 1-gamma, histone H1 linker protein, glyceraldehyde-3-phosphate dehydrogenase and α-tubulin were suitable for SE tissues, while elongation factor 1-gamma and actin were best for the germinative organ tissues. Our work will benefit future studies of gene expression and functional analyses of SE in Liriodendron hybrids. It is also serves as a guide of reference gene selection in early embryonic gene expression analyses for other woody plant species.

scholarly journals Appropriate Reference Genes for RT-qPCR Normalization in Various Organs of Anemone Flaccida Fr. Schmidt at Different Growing Stages

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 459
Author(s):  
Zeying Zhao ◽  
Hanwen Zhou ◽  
Zhongnan Nie ◽  
Xuekui Wang ◽  
Biaobiao Luo ◽  
...  
Keyword(s):  
Reference Genes ◽  
Gene Selection ◽  
Quantitative Polymerase Chain Reaction ◽  
Cultivated Plants ◽  
Gene Expressions ◽  
Natural Habitats ◽  
Triterpenoid Saponins ◽  
Qpcr Normalization ◽  
Suitable Reference

Anemone flaccida Fr. Schmidt is a traditional medicinal herb in southwestern China and has multiple pharmacological effects on bruise injuries and rheumatoid arthritis (RA). A new drug with a good curative effect on RA has recently been developed from the extract of A. flaccida rhizomes, of which the main medicinal ingredients are triterpenoid saponins. Due to excessive exploitation, the wild population has been scarce and endangered in a few of its natural habitats and research on the cultivation of the plant commenced. Studies on the gene expressions related to the biosynthesis of triterpenoid saponins are not only helpful for understanding the effects of environmental factors on the medicinal ingredient accumulations but also necessary for monitoring the herb quality of the cultivated plants. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) as a sensitive and powerful technique has been widely used to detect gene expression across tissues in plants at different stages; however, its accuracy and reliability depend largely on the reference gene selection. In this study, the expressions of 10 candidate reference genes were evaluated in various organs of the wild and cultivated plants at different stages, using the algorithms of geNorm, NormFinder and BestKeeper, respectively. The purpose of this study was to identify the suitable reference genes for RT-qPCR detection in A. flaccida. The results showed that two reference genes were sufficient for RT-qPCR data normalization in A. flaccida. PUBQ and ETIF1a can be used as suitable reference genes in most organs at various stages because of their expression stabilitywhereas the PUBQ and EF1Α genes were desirable in the rhizomes of the plant at the vegetative stage.

scholarly journals Selection and validation of appropriate reference genes for RT-qPCR analysis of flowering stages and different genotypes of Iris germanica L

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yinjie Wang ◽  
Yongxia Zhang ◽  
Qingquan Liu ◽  
Haiying Tong ◽  
Ting Zhang ◽  
...  
Keyword(s):  
Reference Genes ◽  
Molecular Mechanisms ◽  
Gene Selection ◽  
Expression Patterns ◽  
Herbaceous Plant ◽  
Flower Bud ◽  
Perennial Herbaceous Plant ◽  
Iris Germanica ◽  
Accurate Normalization ◽  
Suitable Reference

AbstractIris germanica L. is a perennial herbaceous plant that has been widely cultivated worldwide and is popular for its elegant and vibrantly colorful flowers. Selection of appropriate reference genes is the prerequisite for accurate normalization of target gene expression by quantitative real-time PCR. However, to date, the most suitable reference genes for flowering stages have not been elucidated in I. germanica. In this study, eight candidate reference genes were examined for the normalization of RT-qPCR in three I. germanica cultivars, and their stability were evaluated by four different algorithms (GeNorm, NormFinder, BestKeeper, and Ref-finder). The results revealed that IgUBC and IgGAPDH were the most stable reference genes in ‘00246’ and ‘Elizabeth’, and IgTUB and IgUBC showed stable expression in ‘2010200’. IgUBC and IgGAPDH were the most stable in all samples, while IgUBQ showed the least stability. Finally, to validate the reliability of the selected reference genes, the expression patterns of IgFT (Flowering Locus T gene) was analyzed and emphasized the importance of appropriate reference gene selection. This work presented the first systematic study of reference genes selection during flower bud development and provided guidance to research of the molecular mechanisms of flowering stages in I. germanica.

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