scholarly journals Improved Quantification of DNA Methylation Using Methylation-Sensitive Restriction Enzymes and Real-Time PCR

Epigenetics ◽  
2007 ◽  
Vol 2 (2) ◽  
pp. 86-91 ◽  
Author(s):  
Ko Hashimoto ◽  
Shoichi Kokubun ◽  
Eiji Itoi ◽  
Helmtrud I. Roach
Keyword(s):  
Dna Methylation ◽  
Real Time ◽  
Real Time Pcr ◽  
Restriction Enzymes

scholarly journals Restriction Site–Specific Methylation Studies of Imprinted Genes with Quantitative Real-Time PCR

2008 ◽  
Vol 54 (3) ◽  
pp. 491-499 ◽  
Author(s):  
Sara Bruce ◽  
Katariina Hannula-Jouppi ◽  
Cecilia M Lindgren ◽  
Marita Lipsanen-Nyman ◽  
Juha Kere
Keyword(s):  
Real Time ◽  
Internal Control ◽  
Real Time Pcr ◽  
Uniparental Disomy ◽  
Restriction Enzymes ◽  
Chromosome 7 ◽  
Imprinted Genes ◽  
Quantitative Real Time Pcr ◽  
Large Patient ◽  
The Mean

Abstract Background: Epigenetic studies, such as the measurement of DNA methylation, are important in the investigation of syndromes influenced by imprinted genes. Quick and accurate quantification of methylation at such genes can be of appreciable diagnostic aid. Methods: We first digested genomic DNA with methylation-sensitive restriction enzymes and used DNA without digestion as a control and nonmethylated λ DNA as an internal control for digestion efficiency. We then performed quantitative real-time PCR analyses with 6 unique PCR assays to investigate 4 imprinting control regions on chromosomes 7 and 11 in individuals with uniparental disomy of chromosome 7 (UPD7) and in control individuals. Results: Our validation of the method demonstrated both quantitative recovery and low methodologic imprecision. The imprinted loci on chromosome 7 behaved as expected in maternal UPD7 (100% methylation) and paternal UPD7 (<10% methylation). In controls, the mean (SD) for percent methylation at 2 previously well-studied restriction sites were 46% (6%) for both H19 and KCNQ1OT1, a result consistent with the previously observed methylation rate of approximately 50%. The methylation percentages of all investigated imprinted loci were normally distributed, implying that the mean and SD can be used as a reference for screening methylation loss or gain. Conclusion: The investigated loci are of particular importance for investigating the congenital Silver–Russell and Beckwith–Wiedemann syndromes; however, the method can also be applied to other imprinted regions. This method is easy to set up, has no PCR bias, requires small amounts of DNA, and can easily be applied to large patient populations for screening the loss or gain of methylation.

scholarly journals High-fat diet-induced and genetically inherited obesity differentially alters DNA methylation profile in the germline of adult male rats

2020 ◽  
Vol 12 (1) ◽  
Author(s):  
Sharvari S. Deshpande ◽  
Harishankar Nemani ◽  
Gandhimathi Arumugam ◽  
Avinash Ravichandran ◽  
Nafisa H. Balasinor
Keyword(s):  
Dna Methylation ◽  
Real Time ◽  
Real Time Pcr ◽  
High Fat Diet ◽  
Male Rats ◽  
Global Dna Methylation ◽  
High Fat ◽  
Differential Effects ◽  
Male Germline ◽  
Embryo Loss

Abstract Background Paternal obesity has been associated with reduced live birth rates. It could lead to inheritance of metabolic disturbances to the offspring through epigenetic mechanisms. However, obesity is a multifactorial disorder with genetic or environmental causes. Earlier we had demonstrated differential effects of high-fat diet-induced obesity (DIO) and genetically inherited obesity (GIO) on metabolic, hormonal profile, male fertility, and spermatogenesis using two rat models. The present study aimed to understand the effect of DIO and GIO on DNA methylation in male germline, and its subsequent effects on the resorbed (post-implantation embryo loss) and normal embryos. First, we assessed the DNA methylation enzymatic machinery in the testis by Real-Time PCR, followed global DNA methylation levels in spermatozoa and testicular cells by ELISA and flow cytometry, respectively. Further, we performed Methylation Sequencing in spermatozoa for both the groups. Sequencing data in spermatozoa from both the groups were validated using Pyrosequencing. Expression of the differentially methylated genes was assessed in the resorbed and normal embryos sired by the DIO group using Real-Time PCR for functional validation. Results We noted a significant decrease in Dnmt transcript and global DNA methylation levels in the DIO group and an increase in the GIO group. Sequencing analysis showed 16,966 and 9113 differentially methylated regions in the spermatozoa of the DIO and GIO groups, respectively. Upon pathway analysis, we observed genes enriched in pathways involved in embryo growth and development namely Wnt, Hedgehog, TGF-beta, and Notch in spermatozoa for both the groups, the methylation status of which partially correlated with the gene expression pattern in resorbed and normal embryos sired by the DIO group. Conclusion Our study reports the mechanism by which diet-induced and genetically inherited obesity causes differential effects on the DNA methylation in the male germline that could be due to a difference in the white adipose tissue accumulation. These differences could either lead to embryo loss or transmit obesity-related traits to the offspring in adult life.

Identification of epigenetic silencing of GCNT2 expression by comprehensive real-time PCR screening in colorectal cancer.

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. 506-506
Author(s):  
Kazunorii Nakamura ◽  
Horomichi Sawaki ◽  
Keishi Yamashita ◽  
Masahiko Watanabe ◽  
Hisashi Narimatsu
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Critical Role ◽  
Normal Mucosa ◽  
Primary Cancer ◽  
Normal Tissues ◽  
Cancer Tissues

506 Background: Glycoprotein expression profile has been proved to be dramatically altered in human cancers, however specific glycogenes which are aberrant in expression in cancer cells has not been fully identified. Recent accumulated evidence supported notion that the reduced expression of tumor suppressor genes is explained by DNA promoter methylation in human cancer. Methods: We used Comprehensive Real time PCR system (CRPS) for glycogenes (189 genes) to identify genes aberrantly expressed in colorectal cancer tissues (CRC) as compared to the corresponding normal mucosa tissues. GCNT2 was of particular interest among the identified genes in CRC. Results: (1) GCNT2 harbors 3 isoforms which have different promoter regions. (2) All of the 3 isoforms of GCNT2 genes were remarkably decreased in CRC as compared to the corresponding normal mucosa, and each isoform expression was strongly associated with other 2 isoforms in primary cancer tissues by TaqMan real time PCR (R = 0.99-995, p < 0.0001). (3) Among the 5 CRC cell lines (DLD1, HCT116, CACO2, LOVO), those which were silenced in expression were reactivated by demethylating agents such as 5-aza-2’ deoxycytidine and trichostatin A. (4) Promoter region of the variant 2 of GCNT2 was consistent with its silenced expression in CRC cell lines by cloned sequence, so we examined DNA methylation status of the promoter of the GCNT2 variant 2 in 50 primary cancer tissues and the corresponding normal tissues. Quantitative MSP revealed that almost half of normal tissues have methylation as high as tumor tissues, while, in the primary CRC with less methylation in the corresponding normal tissues, DNA methylation was higher in primary CRC tissues than in the corresponding normal tissues. Finally, GCNT2 variant 2 stable transfection induced expression of other 2 isoform variants. Conclusions: We identified novel methylation gene GCNT2 among the glycoenes. Glycoenes that were altered in genomic or epigenetic manner have been few, so GCNT2 may play a critical role in cancer progression through glycan change.

67 CELL TYPE AND CULTURE OVER TIME EFFECT DNA METHYLTRANSFERASE 1 EXPRESSION IN BOVINE DONOR FIBROBLAST CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 151 ◽  
Author(s):  
K. Moore ◽  
E. Wroclawska ◽  
J. M. Kramer ◽  
S. L. Goicoa
Keyword(s):  
Dna Methylation ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Culture Conditions ◽  
Fibroblast Cells ◽  
Cell Type ◽  
Donor Cells ◽  
Dnmt1 Expression ◽  
Over Time

Aberrant chromatin remodeling has been implicated in the low success rates achieved from cloned embryos. Following fertilization, DNA methylation within a normal embryo is rapidly reduced to a very low level and remains low until the 8–16 cell stage when DNA methylation once again increases. In contrast, the majority of cloned embryos fail to exhibit a similar methylation pattern. This may be due to somatic cell-associated DNMT1s keeping methylation high. However, attempts to chemically modify methylation patterns of donor cells prior to cloning have proven problematic. The objective of this study was to determine if a more natural approach, such as culture conditions, time in culture, and/or cell type, could alter DNMT1 expression in donor fibroblast cells. Two experiments were designed to meet these objectives. Donor fibroblast cell lines were produced from biopsies taken from male and female skin, ovaries, and testes, and were grown in Dulbecco&apos;s modified Eagle&apos;s medium (DMEM) supplemented with 15&percnt; fetal bovine serum, 0.1 mM non-essential amino acids, 2 mM L-glutamine, and 0.1 mM β-mercaptoethanol, in a humidified environment of 5&percnt; CO2 in air, at 39&deg;C. In Experiment 1, cell lines were maintained at 70&percnt; confluence to passage 4, 8, and 12, and analyzed by reverse transcription real-time PCR. In Experiment 2, cell lines were evaluated under 3 culture conditions: proliferating (70&percnt; confluence), serum-starved (0.5&percnt; FBS), and confluent (100&percnt;), and analyzed by reverse transcription real-time PCR. RNA was isolated from cell lines using Trizol reagent (Invitrogen, Carlsbad, CA, USA), reverse transcribed, and analyzed for DNMT1 expression using Taqman real-time PCR, with β-actin as the reference standard. All samples and no template controls were run in triplicate. Final quantitation was done using the comparative CT method, and relative DNMT1 expression was analyzed using one-way ANOVA followed by LS means multiple comparisons. Cell type and passage number had a significant effect on DNMT1 expression. Ovarian fibroblasts had an overall increase in expression in DNMT1 over time (P &lt; 0.05), whereas male skin fibroblasts demonstrated an opposite trend (P &equals; 0.05). Female skin fibroblasts and testes fibroblasts also had a decrease in DNMT1 expression over time, but only approached significance (P &lt; 0.10). For Experiment 2, culture conditions tested did not affect DNMT1 expression for any except one skin cell line. In that case, proliferating cells had significantly higher DNMT1 than quiescent cells (P &lt; 0.005). This research emphasizes the importance of donor cell type and culture effects over time on gene expression. These important aspects should be considered when selecting and growing donor cells to be utilized in somatic cell nuclear transfer. This project was supported by National Research Initiative Competitive Grant no. 2006-35203-16620 from the USDA Cooperative State Research, Education, and Extension Service and the Florida Agricultural Experiment Station.

siRNA Targeting DNA Methyltransferase I Increases ε- and γ-Globin Expression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 973-973
Author(s):  
Virryan Banzon ◽  
Vinzon Ibanez ◽  
Kestis Vaitkus ◽  
Kenneth Peterson ◽  
Joseph DeSimone ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Real Time ◽  
Real Time Pcr ◽  
Dna Methyltransferase ◽  
Globin Gene ◽  
Pcr Analysis ◽  
Globin Promoter ◽  
Chain Synthesis ◽  
Globin Chain Synthesis ◽  
In Cells

Abstract Abstract 973 Pharmacological inhibitors of DNA methyltransferase (DNMT) increase fetal hemoglobin (HbF) levels in experimental primates and patients with sickle cell disease. Therefore we hypothesize that DNMT is directly involved in maintaining repression of the γ-globin gene in adult stage erythroid cells. To test this hypothesis, levels of DNMT1 in mouse chemical-of-dimerization (CID) bone marrow (BM) cells containing the human β-globin gene locus in the context of a yeast artificial chromosome (βYAC) and primary cultured erythroid progenitor cells (EPC) derived from baboon CD34+ BM cells were reduced by treatment with siRNA targeting DNMT1 (siDNMT1) and the effect on globin gene expression determined. Nucleofection conditions that achieved 80-90% transfection efficiency were used to introduce siRNA into CID-dependent mouse βYAC BM cells. Real time PCR analysis showed that expression of DNMT1 was decreased 70-80% in cells treated with siDNMT1 compared to cells transfected with nonsilencing control siRNA while DNMT3A and 3B were not decreased. Results of real time PCR analysis of six independent experiments showed that ε-globin expression was increased 65.3+/−37.8 fold, γ-globin 230.3+/−147.8 fold, and β-globin 6.0+/−3.3 fold in cells treated with siDNMT1 compared to untreated controls, while cells treated with nonsilencing siRNA showed minimal (<2 fold) changes. The difference in ε- and γ-globin expression between cells treated with siDNMT1 and nonsilencing RNA was significant (p<.01). Bisulfite sequence analysis showed that DNA methylation of the ε-globin promoter and γ-globin promoters were reduced to 25.7 and 53.3% dmC, respectively, in cells treated with siDNMT1 compared to 68.8 and 98.8% dmC, respectively, in cells treated with nonsilencing siRNA. Nucleofection of cultured baboon EPC with siDNMT1 or nonsilencing siRNA was performed on d7 and d8 of culture. Transfection efficiencies of 45-50 % were achieved. Expression of DNMT1 was decreased >80% in cells treated with siDNMT1 compared to those treated with nonsilencing siRNA. Real time PCR analysis of duplicate samples showed that γ-globin expression was increased 2.06 and 2.25 fold relative to untreated controls following treatment of cells with siDNMT1 while nonsilencing siRNA had no effect. Expression of ε-globin was increased 35.26 and 25.4 fold relative to untreated controls in cells treated with siDNMT1 while a lesser effect was observed in cells treated with nonsilencing siRNA (7.41 and 8.16 fold). HPLC analysis of biosynthetically radiolabelled globin chains showed that γ-globin chain synthesis (γ/γ+β ratio) was increased in cells treated with siDNMT1 (0.59 and 0.63) compared to cells treated with nonsilencing siRNA (0.41 and 0.37), untreated controls (0.39) and mock-transfected controls (0.43). DNA methylation of 3 CpG residues within the 5' ε-globin promoter region and 5 CpG residues within the 5' γ-globin promoter region was reduced to 52.8 and 45.0% dmC, respectively, in cells treated with siDNMT1, compared to 100 and 85.4% dmC, respectively, in cells treated with nonsilencing siRNA. Our results demonstrate that siDNMT1 reduces DNMT1, reduces levels of DNA methylation of the ε- and γ-globin gene promoters, and increases ε- and γ-globin gene expression and γ-globin chain synthesis in CID-dependent mouse BM cells and in primary baboon EPC cultures derived from CD34+ BM cells. We conclude that DNMT1 is critically involved in the mechanism responsible for repression of γ-globin expression in adult-stage erythroid cells and therefore inhibition of DNMT1 activity by pharmacological inhibitors of DNA methyltransferase likely plays a fundamental role in the ability of these drugs to increase HbF in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Sequence-Specific Histone Methylation Is Detectable on Circulating Nucleosomes in Plasma

2008 ◽  
Vol 54 (7) ◽  
pp. 1125-1131 ◽  
Author(s):  
Ugur Deligezer ◽  
Ebru E Akisik ◽  
Nilgün Erten ◽  
Nejat Dalay
Keyword(s):  
Dna Methylation ◽  
Real Time ◽  
Histone Modifications ◽  
Histone Methylation ◽  
Real Time Pcr ◽  
Chromatin Immunoprecipitation ◽  
Single Copy ◽  
Pcr Analysis ◽  
Alu Elements ◽  
Histone 3

AbstractBackground: Alterations in DNA methylation and histone modifications have been implicated in carcinogenesis. Although tumor-specific alterations in DNA methylation can be detected in the serum and plasma of cancer patients, no data are available on the presence of histone modifications in circulating blood. We investigated whether histone methylation, as a model of histone modifications, is detectable in plasma. Because methylation at histone 3 lysine 9 (H3K9) has been demonstrated to be enriched at sites of repetitive ALU elements, we addressed the specificity of histone-methylation detection and hypothesized that if monomethylated H3K9 (H3K9me1) is detectable in plasma, the concentrations in mononucleosomes and oligonucleosomes would be different. We also analyzed a single-copy gene, CDKN2A.Methods: We enrolled 21 multiple myeloma patients in the study. We used ELISA and real-time PCR analysis to evaluate nucleosomes and cell-free DNA, respectively, as evidence of the presence of histones and associated DNA in circulating blood. H3K9me1 was analyzed by chromatin immunoprecipitation.Results: ELISA and real-time PCR assays indicated the presence of free nucleosomes and DNA in plasma, and the results were quantitatively correlated (P &lt; 0.001). The detection of histone methylation on free nucleosomes was sequence dependent. Fragments representing mono- and oligonucleosomes differed with respect to H3K9me1 concentrations (P = 0.004), in accordance with our hypothesis. In addition, the detection rate and concentrations of H3K9me1 were significantly higher on the fragment covering both mononucleosomes and oligonucleosomes than on the CDKN2A promoter (P &lt; 0.001).Conclusions: If validated in further studies, our findings may be a basis for investigations of cancer-specific alterations in histone modifications in the circulation.

scholarly journals Application of DNA methylation-based markers in identification of mixed body fluid evidences simulating crime scene scenarios

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rania Gomaa ◽  
Lamis Nader ◽  
Jumana Jamal
Keyword(s):  
Dna Methylation ◽  
Real Time ◽  
Real Time Pcr ◽  
Peripheral Blood ◽  
Body Fluid ◽  
Crime Scene ◽  
Mendelian Inheritance ◽  
Tissue Type ◽  
Menstrual Blood ◽  
Transcriptional Level

Abstract Background Epigenetic modifications are heritable and follow a non-mendelian inheritance pattern. They do not alter the DNA sequence but affect the gene expression at the transcriptional level. DNA methylation is one of these epigenetic changes and it is characteristic to each tissue and shows specificity with respect to developmental stage and age. Due to its specificity and reliability, it has emerged as a valuable tool in forensic investigation. Biological samples, such as blood, saliva, semen, or hair found at the crime scene can be used to isolate DNA and study the methylation pattern. Recent developments in molecular biology techniques allowed the study of the effects of methylation in specific tissues. DNA methylation specificity is very intense. These specific markers can be used to identify the tissue type such as blood, saliva, or semen at the crime scene and helps in the identification of the culprit. The present study aimed to validate the use of DNA methylation body fluid-specific markers in the identification of peripheral blood, menstrual blood, and semen. Additionally, it aimed to investigate the potential use of such DNA methylation markers for the identification of different body fluids mixtures simulating forensic science scenarios. Different DNA methylation markers were studied in different body fluid samples (peripheral blood, menstrual blood, and semen) individually and as mixtures. DNA extraction and bisulfite conversion were performed and followed by real-time PCR. Results The results of real-time PCR and the statistical analysis showed that the SPERM2 marker was better than SEU2 in the identification of semen DNA in mixed samples. However, in the identification of individual semen samples, the later marker showed better results than the first one, whereas BLM1 and MENS1 markers were successful in identifying the peripheral and menstrual blood samples, respectively. Conclusions This data can be readily used and applied on different forensic samples for tissue identification. Further sequencing studies are strongly recommended.

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