Quantification of amoA gene abundance and their amoA mRNA levels in activated sludge by real-time PCR

2004 ◽  
Vol 50 (8) ◽  
pp. 1-8 ◽  
Author(s):  
N. Araki ◽  
T. Yamaguchi ◽  
S. Yamazaki ◽  
H. Harada
Keyword(s):  
16S Rrna ◽  
Dissolved Oxygen ◽  
Real Time ◽  
Real Time Pcr ◽  
Ammonia Oxidation ◽  
Amoa Gene ◽  
Ammonia Concentration ◽  
Ammonia Oxidizing Bacteria ◽  
Mrna Levels ◽  
Transcription Level

The transcription level of amoA mRNA encoding a subunit of ammonia monooxygenase (AMO) in ammonia-oxidizing bacteria (AOB) was quantified by reverse transcription-polymerase chain reaction (RT-PCR) methods in combination with real-time PCR technology. The effects of ammonia concentration and dissolved oxygen (DO) on the transcription levels of amoA mRNA and 16S rRNA in AOB were evaluated in batch experiments with nitrifying sludge taken from a lab-scale reactor treating artificial wastewater. A batch incubation without ammonia resulted in a rapid decrease, within four hours, in the transcription level of amoA mRNA to as low as 1/10 of that at the beginning of the experiment, while the 16S rRNA level in AOB was almost constant. After subsequent incubation with 3 mM ammonia for eight hours, a small increase in the transcription level of amoA mRNA occurred, but ammonia oxidation proceeded in the interim. Copy numbers of amoA mRNA showed an almost fixed value for over eight hours in the absence of dissolved oxygen.

Genome-wide screening of m6A-modified transcript in the Wilms’ tumor

2021 ◽  
Author(s):  
Zhuo Liu ◽  
Feng He ◽  
Jing Liu ◽  
Shengrong OuYang ◽  
Zexi Li ◽  
...  
Keyword(s):  
Gene Ontology ◽  
Real Time ◽  
Real Time Pcr ◽  
Wilms Tumor ◽  
Expression Patterns ◽  
Mrna Levels ◽  
Gene Ontology Analysis ◽  
Quantitative Real Time Pcr ◽  
Related Factors ◽  
Significant Difference

Abstract Background Wilms’ tumor, also called nephroblastoma, is the most common pediatric renal malignancy. The pathogenesis of Wilms’ tumor has been attributed to several genetic and epigenetic factors. However, the most pervasive internal mRNA modification that affects almost every process of RNA metabolism, RNA N6-Methyladenosine (m6A) methylation, has not been characterized in Wilms’ tumor. Methods Wilms’ tumor (WT) and adjacent non-cancerous (NC) tissue samples were obtained from 23 children with nephroblastoma, and the global m6A levels were measured by mass spectrometry. Analyses by m6A-mRNA epitranscriptomic microarray and mRNA microarray were performed, and m6A-related mRNAs were validated by quantitative real-time PCR for input and m6A-immunoprecipitated RNA samples from WT and NC tissues. Gene ontology analysis and KEGG pathway analysis were performed for differentially expressed genes, and expression of RNA methylation-related factors was measured by quantitative real-time PCR. Results The total m6A methylation levels in total RNA of WT samples and NC samples were (0.21 ± 0.01)% and (0.22 ± 0.01)%, respectively, with no statistically significant difference. Fifty-nine transcripts were differentially m6A-methylated between the WT and NC groups, which showed distinct m6A modification patterns. Gene ontology analysis indicated that m6A-modified genes were enriched in cancer-associated pathways, including the mTOR pathway, and conjoint analysis of the unique methylation and gene expression patterns in WT samples suggested an association with metabolic pathways.The mRNA levels of the m6A-related “reader” genes, YTHDF1, YTHDF2 and IGF2BP3, were statistically higher in WT samples than in NC samples. Conclusion This is the first study to determine the m6A modification profiles in Wilms’ tumor. Our data provide novel information regarding patterns of m6A modification that correlate with carcinogenesis in Wilms’ tumor.

scholarly journals Cloning, Characterization, and Expression Features of Chicken CDS2 Splicing Variants

2020 ◽  
Author(s):  
Yuanyuan Xu ◽  
Shuping Zhang ◽  
Yujun Guo ◽  
Wen Chen ◽  
Yanqun Huang
Keyword(s):  
Adipose Tissue ◽  
Real Time ◽  
Real Time Pcr ◽  
Expression Patterns ◽  
Mrna Levels ◽  
Exogenous Insulin ◽  
Quantitative Real Time Pcr ◽  
Temporal Expression ◽  
Spatio Temporal ◽  
The Brain

Abstract Background: The CDS gene encodes the CDP-diacylglycerol synthase enzyme that catalyzes the formation of CDP-diacylglycerol (CDP-DAG) from phosphatidic acid. At present, there are no reports of CDS2 in birds. Here, we identified chicken CDS2 transcripts by combining conventional RT- PCR amplification, 5' RACE (Fig. 1A), and 3' RACE, explored the spatio-temporal expression profiles of total CDS2 and the longest transcript variant CDS2-4, and investigated the effect of exogenous insulin on total the mRNA level of CDS2 by quantitative real-time PCR. Results: Four transcripts of chicken CDS2 (CDS2-1, -2, -3, and -4) were identified, which were alternatively spliced at the 3′-untranslated region (UTR). CDS2 was widely expressed in all tissues examined and the longest variant CDS2-4 was the major transcript. Both total CDS2 and CDS2-4 were prominently expressed in adipose tissue and the heart, and exhibited low expression in the liver and pectoralis of 49 day-old chickens. Quantitative real-time PCR revealed that total CDS2 and CDS2-4 had different spatio-temporal expression patterns in chicken. Total CDS2 exhibited a similar temporal expression tendency with a high level in the later period of incubation (embryonic day 19 [E19] or 1-day-old) in the brain, liver, and pectoralis. While CDS2-4 presented a distinct temporal expression pattern in these tissues, CDS2-4 levels peaked at 21 days in the brain and pectoralis, while liver CDS2-4 mRNA levels were highest at the early stage of hatching (E10). Total CDS2 (P < 0.001) and CDS2-4 (P = 0.0090) mRNA levels in the liver were differentially regulated throughout development of the chicken. Exogenous insulin significantly downregulated the level of total CDS2 at 240 min in the pectoralis of Silky chickens (P < 0.01). Total CDS2 levels in the liver of Silky chickens were higher than that of the broiler in the basal state and after insulin stimulation. Conclusion: Chicken CDS2 has multiple transcripts with variation at the 3′-UTR, which was prominently expressed in adipose tissue. Total CDS2 and CDS2-4 presented distinct spatio-temporal expression patterns, and they were differentially regulated with age in liver. Insulin could regulate chicken CDS2 levels in a breed- and tissue-specific manner.

Investigation of the effect of Prunus Amygdalus Amara on the expression of some genes of apoptosis and immortality in breast cancer cells (MCF-7)

2021 ◽  
Vol 13 ◽  
Author(s):  
Maryam Abdolahi-Majd ◽  
Gholamhossein Hassanshahi ◽  
Mahboubeh Vatanparast ◽  
Mojgan Noroozi Karimabad
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Prunus Amygdalus ◽  
Mcf7 Cells ◽  
Significant Difference ◽  
Anti Cancer ◽  
Bitter Almond ◽  
Mcf 7

Background: Anti-cancer effects of almond nuts or oil have been approved, but there are a few pieces of research that have evaluated, in detail, almond and other seeds' effects on cancer. Therefore, in the present project, the aim was to explore the regulatory effect of the bitter almond extract (Prunus amygdalus Batsch) on the apoptotic and anti-cancer potency of MCF-7 cells. Objectives: In the current experimental research, the Almond effect on MCF7 cells was evaluated by investigating the expression and the balance between Bcl-2, Bax genes to unmark the potential molecular mechanism. Methods: For 24 and 48h, the MCF7 cells were treated with the bitter almond extract (187.5-3000 µg/mL). MTT assay was used to assess the viability, and Real-time-PCR was applied to determine the expression of Bax and Bcl-2, facing β-actin. Results: Our results revealed a significant difference between different extract concentrations on the viability of MCF7 cell lines in 24 and 48 h; cell viability decreased time-dependently (P < 0.05). After 24 and 48h of extract facing MCF7 cells, the evaluated IC50 value was 3000 and 1500 µg/mL, respectively. Based on Real Time-PCR analysis, after 24 and 48 h, the mRNA levels of BCL-2 decreased by the extract, whereas BAX was in the MCF-7 cell line. Conclusion: From the results, it can be concluded that bitter almond extract has anti-cancer properties that may influence the apoptotic pathways by regulating relative gene expression.

Abstract 1064: Pellino-1: A Novel Candidate In Vegf/Flk-1/MKK2 Signaling In Ischemic Preconditioning Induced Cardioprotection: A Study Using Flk +/− and MKK2 −/− Knockout Mice.

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Mahesh Thirunavukkarasu ◽  
Samson Mathews Samuel ◽  
Sankar Addya ◽  
Lijun Zhan ◽  
Chi-Kuang Huang ◽  
...  
Keyword(s):  
Real Time ◽  
Ischemic Preconditioning ◽  
Knockout Mice ◽  
Real Time Pcr ◽  
Left Ventricular ◽  
List Type ◽  
Mrna Levels ◽  
Clinical Issue ◽  
Endothelial Cell Function ◽  
Vegf Signaling

VEGF modulates the complex process of angiogenesis and other various aspects of endothelial cell function through either of its two tyrosine kinase receptors, VEGFR1/Flt-1 or VEGFR2/Flk1/ KDR via its target protein MKK2. In the present study we used Flk1 +/− and MKK2 −/− knockout mice in an attempt to address an important clinical issue by identifying potential downstream candidates of VEGF signaling through Flk1 receptor that trigger cardioprotective signal during ischemic preconditioning (IP). Mouse hearts were subjected to 30 min of global ischemia and 2 hours of reperfusion (IR) in the Isolated Working Heart model. It is known that IP (4min of ischemia + 6min reperfusion, 4 cycles, before 30 min of ischemia) induces cardioprotection through the activation of the VEGF signaling cascade. The mice were randomly divided into 6 groups for both the gene knockout (KO) studies: Wild Type-Baseline (WTBL), FlkBL/ MKK2BL (KOBL), WTIR , KOIR, WTIP and KOIP. Significant reduction in left ventricular functional recovery through out reperfusion (dp/dt = 605 vs 884), diminished coronary flow (1.9 vs 2.4) and aortic flow (0.16 vs 1.2) and increased infarct size (38.4% vs. 28.41%) after reperfusion were observed in FlkIR, compared to WTIR. As expected we observed disruption in IP induced cardioprotection in FlkIP compared to WTIP. Affymetrix gene chip analysis demonstrated significant downregulation of genes (Pellino-1, MKK2, NF-ΰB) which are thought to play important roles in cardioprotection after ischemic insults in the Flk +/− mice compared to WT. These results were further validated at the mRNA expression level with Real Time PCR. Pellino-1 (Pel-1) was found to be significantly downregulated in FlkBL (0.74 vs 1), FlkIR (1.29 vs 1.35) and FlkPC (1.35 vs 1.49) as compared to their respective controls. We further validated the mRNA levels of Pel-1using Real Time PCR and RT-PCR in the MKK2 −/− mice and found that it, remained unaffected in MKK2BL as compared to its WTBL, and increased as expected in MKK2PC as compared to both MKK2BL and FlkPC (2.48 vs 1.2 and 2.48 vs 1.35, respectively). Therefore this study validated for the first time that Pel-1 is a novel downstream mediator in VEGF/FLK1 signaling and it induces IP mediated cardioprotection via MKK2 signaling.

Determining Viability of M. ulcerans by 16S rRNA RT Reverse Transcriptase Real-Time PCR

2021 ◽  
pp. 81-86
Author(s):  
Marcus Beissner ◽  
Richard Odame Phillips ◽  
Gisela Bretzel
Keyword(s):  
16S Rrna ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Real Time Pcr

scholarly journals Quantitative Real-time PCR for the Measurement of 11β-HSD1 and 11β-HSD2 mRNA Levels in Tissues of Healthy Dogs

2007 ◽  
Vol 39 (8) ◽  
pp. 548-554 ◽  
Author(s):  
N. Sieber-Ruckstuhl ◽  
M. Meli ◽  
F. Boretti ◽  
E. Gönczi ◽  
H. Lutz ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Mrna Levels ◽  
Quantitative Real Time Pcr ◽  
Healthy Dogs

Monitoring of Microbial Community Inhabiting a Low-Grade Copper Sulphide Ore by Quantitative Real-Time PCR Analysis of 16S rRNA Genes

2007 ◽  
Vol 20-21 ◽  
pp. 539-542 ◽  
Author(s):  
Francisco Remonsellez ◽  
F. Galleguillos ◽  
Sonestie Janse van Rensburg ◽  
G.F. Rautenbach ◽  
Pedro A. Galleguillos ◽  
...  
Keyword(s):  
16S Rrna ◽  
Real Time ◽  
Real Time Pcr ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Low Grade ◽  
Copper Sulphide ◽  
Quantitative Real Time Pcr ◽  
Sulphide Ore ◽  
Heap Bioleaching

Microbial heap bioleaching is being used as an industrial process to recover copper from low grade ores. It is known that a consortium of different microorganisms participates in this process. Therefore identification and quantification of communities inhabiting heap bioleaching operations is a key step for understanding the dynamics and role of these microorganisms in the process. A quantitative real-time PCR approach was used to investigate the microbial dynamics in this process. To study the microbial population inhabiting a low-grade copper sulphide ore bioleaching industrial heap process at Escondida Mine in Chile, 16S rRNA genetic libraries were constructed using bacterial and archaeal universal primers. Phylogenetic analyses of sequences retrieved from genetic libraries showed that the community is mainly composed by microoganisms related to Acidithiobacillus ferrooxidans (2 strains), Acidithiobacillus thiooxidans, Leptospirillum ferrooxidans, Leptospirillum ferriphilum and the archaea Ferroplasma. Specific primers for real-time PCR determination were designed and tested to amplify each of the sequences obtained by cloning. Standard curves for real time PCR were performed using plasmid DNA from selected clones. This methodology is actually being used to monitor relevant microorganisms inhabiting this low-grade copper sulphide ore bioleaching industrial heap.

p29ING4 Regulates the Production of Pro-Angiogenic Molecules by Human Myeloma Cells in Normoxic and Hypoxic Conditions Being Involved in Myeloma-Induced Angiogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 515-515
Author(s):  
Sara Tagliaferri ◽  
Francesca Morandi ◽  
Paolo Lunghi ◽  
Simona Colla ◽  
Mirca Lazzaretti ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Mrna Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Interleukin 8 ◽  
Mrna Levels ◽  
Quantitative Real Time Pcr ◽  
Angiogenic Switch ◽  
Hypoxic Conditions

Abstract Multiple myeloma (MM) cells produce several angiogenic molecules as VEGF, Angiopoietin-1 (Ang-1), interleukin-8 (IL-8) and osteopontin (OPN), however the molecular mechanisms underlying the angiogenic switch are not completely elucidated. The candidate tumor suppressor gene inhibitor of growth family member 4 (p29ING4) has been recently implicated in solid tumors as a repressor of angiogenesis and tumor growth through the suppression of angiogenic related molecules including interleukin-8 (IL-8) and the hypoxia inducible factor (HIF)-1 alpha. In this study we investigate the potential involvement of p29ING4 in the angiogenic switch in MM. First using quantitative real time PCR we compared p29ING4 with VEGF, Ang-1, IL-8 and OPN mRNA levels in eight human myeloma cell lines (HMCLs). A significantly negative correlation was observed between ING4 and IL-8 and a trend of correlation with OPN. Following we transfected HMCLs JJN3, OPM-2 and RPMI-8226 with specific siRNA to completely block the expression of p29ING4 checking the effect on the expression and production of the myeloma-related angiogenic molecules VEGF, Ang-1, IL-8 and OPN by quantitative real time PCR and ELISA assay. p29ING4 suppression in HMCLs did not affect VEGF and Ang-1 production but induced a strong up-regulation of IL-8 mRNA and IL-8 protein secretion. Similarly an induction of OPN mRNA expression as well as OPN secretion was induced by siRNA anti-p29ING4. Moreover conditioned media of HMCLs transfected with siRNA anti p29ING4 significantly increased vessel formation in an experimental in vitro model of angiogenesis (ANGIO kit) as compared to controls. Further we investigate the role of p29ING4 in the production of angiogenic molecule by MM cells in hypoxic condition compared to normoxic one as well as its potential relationship with HIF-1alpha system. Hypoxia induced HIF-1alpha expression at nuclear level and its activity in HMCLs and p29ING4 suppression by siRNA further induced HIF-1alpha transcriptional activity with an increase of its target gene Nip-3 in HMCLs. In turn the block of HIF1-alpha by specific siRNA up-regulated p29ING4 and suppressed IL-8 and OPN mRNA expression by HMCLs suggesting a relationship between p29ING4 and HIF-1alpha activity. These in vitro observations have been extended in vivo by the finding of a significant correlation between bone marrow (BM) plasma IL-8 levels and p29ING4 mRNA expression in purified MM cells obtained from 40 newly diagnosed MM patients (R=−0.58 Spearman’s 2-tailed test: p=0.04), consistently MM patients with higher BM IL-8 levels have a significantly lower p29ING4 mRNA levels. Similarly MM patients positive for OPN expression with high OPN BM levels had a significant lower p29ING4 levels (p=0.02). Finally we found that MM patients with high microvalscular density (MVD&gt;30) have significant lower p29ING4 levels as compared to those with low MVD (&lt;30) (p=0.04) and that MM patients with histological high grade had significant lower p29ING4 mRNA levels (Mann-Whitney 2-tailed: p=0.05). In conclusion, our data indicate that the tumor suppressor p29ING4 regulate the production of angiogenic molecules by MM cells both in normoxic and hypoxic conditions being involved in MM-induced angiogenesis and potentially in tumor progression.

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