324 EFFECT OF AGING ON AMOUNTS OF DNA METHYLTRANSFERASE mRNA IN MOUSE SPERMATOZOA

2007 ◽  
Vol 19 (1) ◽  
pp. 277
Author(s):  
H. Kato ◽  
T. Koda ◽  
M. Kishimoto ◽  
T. Mitani ◽  
K. Matsumoto ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Methyltransferase ◽  
Physiological Role ◽  
Mrna Levels ◽  
Male Mice ◽  
Expression Levels ◽  
Young Males ◽  
Male Age

The spermatozoon is a specially differentiated cell designed to carry a haploid male genome into an oocyte at fertilization. It recently was reported that a matured spermatozoon contains several kinds of mRNAs and these are delivered into the oocyte at fertilization (Ostermeier et al. 2004 Nature 429, 154). The physiological role of paternally derived mRNAs is not clear; however, there is a report that the DNA methyltransferase (Dnmt) mRNA level in spermatozoa from male rats exposed to ethanol was significantly reduced (Bielawski et al. 2002 Alcohl. Clin. Res. 26, 347–351). The reduction of mRNA levels of Dnmt in spermatozoa would lead to altered epigenetic modification of the genome. Because factors such as age may affect spermatozoa mRNA levels, this study evaluated the effect of individual aging on the expression levels of Dnmts during spermatogenesis. This was accomplished by determining expression levels of Dnmts in the whole testis and in spermatozoa from young and aged mice by quantitative reverse-transcription-PCR. Seven- (young) and 68- (aged) week-old C57BL/6N male mice (n = 3/group) were sacrificed by cervical dislocation and whole testes and matured spermatozoa were collected. Total RNA was extracted and purified from each sample. In this study, 5 Dnmts (Dnmt1s, Dnmt1p, Dnmt3a, Dnmt3b, and Dnmt3l) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a reference gene, were examined for expression levels in whole testis and spermatozoa using SYBR Premix Ex Taq (Takara Bio, Inc., Otsu, Shiga, Japan) and the 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Real-Time PCR runs for each Dnmt and GAPDH were repeated 3 times using different RNA batches from different individuals. The GAPDH expression level was used to normalize the expression levels of each Dnmt. Data were analyzed by Student's t-test. Relative expression levels of each Dnmt in testis from aged males compared to that of young males were 0.94, 1.15, 0.91, 1.15, and 1.14 (Dnmt1s, Dnmt1p, Dnmt3a, Dnmt3b, and Dnmt3l, respectively). There was no difference in the expression levels of the 5 Dnmts examined between testes from aged and young males. On the other hand, the relative amounts of each Dnmt mRNA in spermatozoa from aged males compared to that of young males were 0.87, 0.01, 0.54, 1.07, and 1.75 (Dnmt1s, Dnmt1p, Dnmt3a, Dnmt3b, and Dnmt3l, respectively). There was a significant reduction (P < 0.05) in the amount of Dnmt1p mRNA. The reason why the amount of Dnmt1p mRNA in spermatozoa from aged male mice showed such reduction is not clear. There was no difference in the relative expression levels of Dnmt1p in testis irrespective of male age. Dnmt1p is only translated in the spermatocyte during the pachytene stage in meiosis and its physiological role is not clear. To elucidate this male, age-related reduction of the amount of Dnmt1p mRNA in spermatozoa would clarify part of physiological role of Dnmt1p. This work was supported by Wakayama Prefecture Collaboration of Regional Entities for the Advanced of Technological Excellence, Japan, and by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan.

scholarly journals Simultaneous Quantitative Detection of Relevant Biomarkers in Breast Cancer by Quantitative Real-Time PCR

2006 ◽  
Vol 21 (1) ◽  
pp. 30-39 ◽  
Author(s):  
M. Labuhn ◽  
V. Vuaroqueaux ◽  
F. Fina ◽  
A. Schaller ◽  
I. Nanni-Metellus ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Mrna Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Detection ◽  
Mrna Levels ◽  
Quantitative Real Time Pcr ◽  
Expression Levels ◽  
Qrt Pcr ◽  
Mrna Expression Levels

The assessment of ERα, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERα, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERα, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

Identifying the etiologic role of Parvovirus B19 in non-immune hydrops fetalis by histopathology, immunohistochemistry and nucleic acid testing: a retrospective study

Open Medicine ◽  
2007 ◽  
Vol 2 (3) ◽  
pp. 271-279 ◽  
Author(s):  
Koray Ergunay ◽  
Gulcin Altinok ◽  
Bora Gurel ◽  
Ahmet Pinar ◽  
Arzu Sungur ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
San Francisco ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Parvovirus B19 ◽  
Etiologic Agent ◽  
Hydrops Fetalis ◽  
Nucleic Acid Detection

AbstractIntrauterine Parvovirus B19 infections may cause fetal anemia, non-immune hydrops fetalis or abortion. This study focuses on the pathogenic role of Parvovirus B19 in non-immune hydrops fetalis at Hacettepe University, a major reference hospital in Turkey. Twenty-two cases of non-immune hydrops fetalis were retrospectively selected out of a total of 431 hydrops fetalis specimens from the Department of Pathology archieves. Paraffine embedded tissue sections from placental and liver tissues from each case were evaluated by histopathology, immunohistochemistry, nested PCR and commercial quantitative Real-time PCR. Viral DNA was detected in placental tissues by Real-time PCR in 2 cases (2/22, 9.1%) where histopathology also revealed changes suggestive of Parvovirus B19 infection. No significant histopathologic changes were observed for the remaining sections. Nested PCR that targets the VP1 region of the viral genome and immunohistochemistry for viral capsid antigens were negative for all cases. As a result, Parvovirus B19 is identified as the etiologic agent for the development of non-immune hydrops fetalis for 9.1% of the cases in Hacettepe University, Turkey. Real-time PCR is observed to be an effective diagnostic tool for nucleic acid detection from paraffine embedded tissues. Part of this study was presented as a poster at XIIIth International Congress of Virology, San Francisco, USA (Abstract V-572).

scholarly journals Quantitative assessment of CYP11B1 and CYP11B2 expression in aldosterone-producing adenomas

2002 ◽  
pp. 795-802 ◽  
Author(s):  
F Fallo ◽  
V Pezzi ◽  
L Barzon ◽  
P Mulatero ◽  
F Veglio ◽  
...  
Keyword(s):  
Real Time ◽  
Secretory Activity ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Pathophysiological Role ◽  
Aldosterone Production

BACKGROUND: The presence and pathophysiological role of CYP11B1 (11beta-hydroxylase) gene in the zona glomerulosa of human adrenal cortex is still controversial. METHODS: In order to specifically quantify CYP11B1, CYP11B2 (aldosterone synthase) and CYP17(17alpha-hydroxylase) mRNA levels, we developed a real-time RT-PCR assay and examined the expression in a series of adrenal tIssues, including six normal adrenals from patients adrenalectomized for renal cancer and twelve aldosterone-producing adenomas (APA) from patients with primary aldosteronism. RESULTS: CYP11B1 mRNA levels were clearly detected in normal adrenals, which comprised both zona glomerulosa and fasciculata/reticularis cells, but were also measured at a lower range (P<0.05) in APA. The levels of CYP11B2 mRNA were lower (P<0.005) in normal adrenals than in APA. CYP17 mRNAlevels were similar in normal adrenals and in APA. In patients with APA, CYP11B2 and CYP11B1 mRNA levels were not correlated either with basal aldosterone or with the change from basal aldosterone in response to posture or to dexamethasone. No correlation between CYP11B1 mRNA or CYP11B2 mRNA and the percentage of zona fasciculata-like cells was observed in APA. CONCLUSIONS: Real-time RT-PCR can be reliably used to quantify CYP11B1 and CYP11B2 mRNA levels in adrenal tIssues. Expression of CYP11B1 in hyperfunctioning zona glomerulosa suggests an additional formation of corticosterone via 11beta-hydroxylase, providing further substrate for aldosterone biosynthesis. CYP11B1 and CYP11B2 mRNA levels in APA are not related to the in vivo secretory activity of glomerulosa cells, where post-transcriptional factors might ultimately regulate aldosterone production.

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.

scholarly journals Potential prognostic value of PD-L1 and NKG2A expression in Indonesian patients with skin nodular melanoma

2021 ◽  
Author(s):  
Ridwan Dwi Saputro ◽  
Hanggoro Tri Rinonce ◽  
Yayuk Iramawasita ◽  
Muhammad Rasyid Ridho ◽  
Maria Fransiska Pudjohartono ◽  
...  
Keyword(s):  
Target Genes ◽  
Therapy Response ◽  
Mrna Levels ◽  
Independent Predictive Factor ◽  
Clinicopathologic Characteristics ◽  
Tissue Samples ◽  
Nodular Melanoma ◽  
Expression Levels ◽  
Melanoma Tissue

Abstract Objective Biomarker mRNA levels have been suggested to be predictors of patient survival and therapy response in melanoma cases. This study aimed to investigate the correlations between the mRNA expression levels of PD-L1 and NKG2A in melanoma tissue and clinicopathologic characteristics and survival in Indonesian patients with primary nodular melanoma. Results Thirty-two tissue samples were analyzed. Upregulated PD-L1 was associated with shorter overall survival (hazard ratio: 2.930; 95% confidence interval: 1.011–8.489, p = 0.048) compared with patients with normoregulated PD-L1. A significant positive correlation was found between the expression levels of PD-L1 and NKG2A (rs: 0.768, p < 0.001). However, no clinicopathologic associations with PD-L1 and NKG2A mRNA levels were statistically proven. Comparison with other studies suggested that the choice of adjuvant therapy and the presence of TILs affect the prognostic role of PD-L1 expression. NKG2A was not proven to be an independent predictive factor but may become an adjunct target for therapy. The strong correlation between PD-L1 and NKG2A suggests that anti-PD-1 and anti-NKG2A agents could be effective in patients with PD-L1 upregulation. The combination of the mRNA levels of these two target genes may provide a novel prognostic and therapeutic direction for immunotherapy.

scholarly journals Changes in the expression of galanin and galanin receptors in the wall of the colon in pigs experimentally infected with Brachyspira hyodysenteriae

2014 ◽  
Vol 58 (1) ◽  
pp. 23-28 ◽  
Author(s):  
Krzysztof Wąsowicz ◽  
Piotr Podlasz ◽  
Małgorzata Chmielewska ◽  
Katarzyna Łosiewicz ◽  
Jerzy Kaleczyc ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Experimental Colitis ◽  
Expression Level ◽  
Colonic Wall ◽  
Brachyspira Hyodysenteriae ◽  
Expression Levels ◽  
Gapdh Expression ◽  
Galanin Receptors ◽  
Different Levels

Abstract The expression of galanin (GAL) and its three receptors (GalR1, GalR2, and GalR3) were studied with real-time PCR in the colonic wall of pigs suffering from experimental colitis caused by the infection with Brachyspira hyodysenteriae. The expression was studied in the muscular membrane, mucosa/submucosa layer, and in lymphocytes isolated from mucosa/submucosa. The expression levels were normalized to glyceraldehyde-6-phosphate dehydrogenase (GAPDH) expression and compared to expression levels in control animals. GAL expression was found in all three studied compartments of the colonic wall. A significant decrease in GAL expression level was found in the mucosa/submucosa and in isolated lymphocytes, whereas the decrease was much less profound in the muscular membrane. In the case of galanin receptors their expression was found in all studied compartments of the colonic wall, however at different levels, as compared to GAPDH expression. The decrease of galanin receptors expression was found in all studied compartments of the colonic wall of the sick animals.

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.

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