• Digital PCR
• High-Resolution Melting Curve analysis
• Methylation-Sensitive HRM analysis
• Molecular diagnosis
• Quantitative PCR

• DNA Methylation
• Humans
• Real-Time Polymerase Chain Reaction/methods
• Nucleic Acid Denaturation
• Semiconductors
• Transition Temperature
• Reproducibility of Results

• Clinical application
• Colorectal cancer (CRC)
• CpG islands (CGIs)
• DNA methylation (DNAm)
• Mechanisms

• Humans
• Colorectal Neoplasms/genetics
• Colorectal Neoplasms/pathology
• Colorectal Neoplasms/diagnosis
• DNA Methylation
• Epigenesis, Genetic
• Biomarkers, Tumor/genetics
• Biomarkers, Tumor/metabolism
• Prognosis
• Gene Expression Regulation, Neoplastic
• Clinical Relevance

• APP
• Aβ
• DNA methylation
• histone modification
• tau

• Alzheimer Disease/genetics
• Alzheimer Disease/metabolism
• Alzheimer Disease/pathology
• Humans
• DNA Methylation/genetics
• Epigenesis, Genetic
• Histone Code/genetics
• Histones/metabolism
• Animals

• Pregnancy
• Female
• Animals
• Elephants/genetics
• DNA Methylation
• Reproduction
• Linear Models
• Labor, Obstetric
• Animals, Zoo

• Japan Society for the Promotion of Science
• Environmental Restoration and Conservation Agency
• Japan Science and Technology Agency

• Biomarkers
• Methylation
• Tissue and organ damage
• cfDNA

• Humans
• Cell-Free Nucleic Acids/genetics
• DNA Methylation
• Biomarkers
• Epigenesis, Genetic
• Disease Progression
• Biomarkers, Tumor/genetics

• National Key Research and Development Program of China

• CDKN2B/P15
• Gastric cancer
• Joint effects
• Methylation
• Susceptibility

• LH2020H020 Natural Science Foundation of Heilongjiang Province

Pyrosequencing is one of the technologies widely used for quantitative methylation assessment. The protocol of pyrosequencing experiment consists of PCR amplification of a locus of interest and subsequent sequencing via synthesis of the amplified PCR product. As the PCR in this protocol utilizes one primer set for the amplification of a template originating from both methylated and non-methylated versions of the analysed locus, the unequal amplification of one of the templates may affect the methylation level assessment by pyrosequencing. We have investigated whether the unequal amplification of one of the templates challenges the quantitative properties of the pyrosequencing technology. Our results show that the sensitivity and dynamic range of pyrosequencing can be significantly affected by unequal amplification of the methylated and non-methylated version of the locus of interest in an assay specific manner. Thus, the assessment of the effect of unequal template amplification on the performances of the specific pyrosequencing assay is necessary before using the assay either in research or especially in diagnostic settings.

• Bisulfite
• DNA Methylation Sequencing
• High resolution melting
• Validation

• DNA Methylation/genetics
• Genomics
• High-Throughput Nucleotide Sequencing
• Polymerase Chain Reaction/methods
• Sequence Analysis, DNA/methods

Extramammary Paget's disease (EMPD) is a rare skin tumor. Hypermethylation in the MSH2 promoter resulting in the downregulation of its protein expression shows a high detection rate in EMPD tumor tissue, which indicates that the methylation of MSH2 may play an important role in the pathogenesis of EMPD.

This study aims to establish a rapid analysis strategy based on the methylation-sensitive high-resolution melting curve (MS-HRM) to detect the methylation level of the MSH2 promoter.

With the use of universal methylated human DNA products, we established the MS-HRM standard curve to quantitatively detect the methylation level of the MSH2 promoter. Then, all 57 EMPD tumor DNA samples were analyzed. Pyrosequencing assay was also carried out to test the accuracy and efficacy of MS-HRM. Besides, a total of 54 human normal and other cancerous tissues were included in this study to test the reliability and versatility of the MS-HRM standard curve.

In this study, by using the established MS-HRM, we found that 96.5% (55/57) EMPD tumor samples had varying methylation levels in the MSH2 promoter ranging from 0% to 30%. Then, the methylation data were compared to the results obtained from pyrosequencing, which showed a high correlation between these two techniques by Pearson's correlation (r = 0.9425) and Bland–Altman plots (mean difference = −0.1069) indicating that the methylation levels analyzed by MS-HRM were consistent with DNA pyrosequencing. Furthermore, in 23 normal and 31 other cancerous tissue samples, there were two colorectal cancer (CRC) tissues that tested MSH2 methylation positive (1% and 5%) which confirmed that our established MS-HRM can be widely applied to various types of samples.

MS-HRM standard curve can be used for the detection of the methylation level of MSH2 in EMPD tumor samples and other cancerous tissues potentially, which presents a promising candidate as a quantitative assay to analyze MSH2 promoter methylation in routine pathological procedure.

• cancer
• disease
• dna methylation
• dysbiosis
• epigenetics
• epigenome
• gut
• inflammatory bowel disease
• microbiome
• necrotizing enterocolitis

• CASK
• colorectal cancer
• dMS-HRM
• heterogeneous methylation
• prognosis

• Colorectal Neoplasms/genetics
• Colorectal Neoplasms/mortality
• DNA Methylation/physiology
• Female
• Guanylate Kinases/genetics
• Humans
• Male
• Middle Aged
• Prognosis
• Survival Rate

• DNA damage
• H3K36me3
• LINE-1 methylation
• MGMT methylation
• occupational PAH exposure

• Body fluid identification
• DNA methylation
• Forensic science
• Genetic variation
• Heritability
• Mutations

Colorectal cancer (CRC) remains a major contributor to the number of cancer-related deaths that occur annually worldwide. With the development of molecular biology methods, an increasing number of molecular biomarkers have been identified and investigated. CRC is believed to result from an accumulation of epigenetic changes, and detecting aberrant DNA methylation patterns is useful for both the early diagnosis and prognosis of CRC. Numerous studies are focusing on the development of DNA methylation detection methods or DNA methylation panels. Thus, this review will discuss the commonly used techniques and technologies to evaluate DNA methylation, their merits and deficiencies as well as the prospects for new methods.

• Colorectal cancer
• Cancer screening
• Marker
• DNA methylation detection

• BRCA1 gene
• TNBC
• methylation in peripheral blood

Aberrant DNA methylation patterns are common in cancers and environmental pollutant exposed subjects. Up to date, few studies have examined the aberrant DNA methylation patterns in benzene exposed workers. We recruited 141 benzene-exposed workers, including 83 benzene-exposed workers from a shoe factory in Wenzhou and 58 workers from a painting workshop in Wuhu, 35 workers in Wuhu were followed from 2009 to 2013, and 48 indoor workers as controls from Wenzhou. We used high-resolution melting (HRM) to quantitate human samples of DNA methylation in long interspersed nuclear element-1 (LINE-1), (6)-methylguanine-DNA methyltransferase (MGMT), and DNA mismatch repair gene human mutator L homologue 1 (hMLH1). AML-5 cells were treated with benzoquinone (BQ) and hydroquinone (HQ), and the promoter methylation of MGMT and hMLH1 was detected using the bisulfite sequencing PCR method. The degree of LINE-1 methylation in benzene-exposed workers was significantly lower than that of the controls (p<0.001), and the degree of MGMT (p<0.001) and hMLH1 (p = 0.01) methylation was significantly higher than that of the controls. The in vitro study validated the aberrant hypermethylation of hMLH1 after treatment with BQ. Among the cohort workers who were followed from 2009 to 2013, the LINE1 methylation elevated in 2013 than 2009 (p = 0.004), and premotor methylation in hMLH1 reduced in 2013 than 2009 (p = 0.045) with the reduction of the benzene exposure. This study provides evidence that benzene exposure can induce LINE-1 hypomethylation and DNA repair gene hypermethylation.

• Adolescent
• Adult
• Aged
• Benzene/adverse effects
• Cell Line
• DNA Methylation/drug effects
• Environmental Pollutants/adverse effects
• Female
• Humans
• Long Interspersed Nucleotide Elements/drug effects
• Male
• Middle Aged
• MutL Protein Homolog 1/genetics
• O(6)-Methylguanine-DNA Methyltransferase/genetics
• Occupational Exposure/adverse effects
• Occupational Exposure/analysis
• Promoter Regions, Genetic/drug effects
• Young Adult

• National Natural Science Foundation of China
• the Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine of China

• Cell-free DNA
• DNA methylation
• Differential biomarker
• MS-HRM
• Misdiagnosis

• Area Under Curve
• Bile Duct Diseases/diagnosis
• Bile Duct Diseases/genetics
• Bile Duct Neoplasms/diagnosis
• Bile Duct Neoplasms/genetics
• Biomarkers, Tumor/genetics
• Cell Adhesion Molecules/genetics
• Cell-Free Nucleic Acids
• Cholangiocarcinoma/diagnosis
• Cholangiocarcinoma/genetics
• DNA Methylation
• Diagnosis, Differential
• Female
• GPI-Linked Proteins/genetics
• Homeodomain Proteins/genetics
• Humans
• Male
• Neoplasm Proteins/genetics
• Sensitivity and Specificity

M1 and M2 macrophage balance in atherosclerosis has attracted much interest. Though, it remains unknown how macrophage heterogeneity is regulated. Moreover, the regulation of macrophage polarization and activation also involve DNA methylation. However, it remains ambiguous which genes are under direct regulation by DNA methylation. Our aim was to evaluate the gene-specific promoter DNA methylation status of M1/M2 polarization markers in PBMCs of CAD patients. A case-control study was performed with 25 CAD patients and 25 controls to study the promoter DNA methylation status of STAT1, STAT6, MHC2, IL12b, iNOS, JAK1, JAK2 and SOCS5 using MS-HRM analysis. Our data indicates that there was a clear-cut difference in the pattern of gene-specific promoter DNA methylation of CAD patients in comparison to controls. A significant difference was observed between the percentage methylation of STAT1, IL12b, MHC2, iNOS, JAK1 and JAK2 in CAD patients and control subjects. In conclusion, our data show that MS-HRM assay is a rapid and inexpensive method for qualitatively identifying aberrant gene-specific promoter DNA methylation changes in CAD. Furthermore, we propose that gene-specific promoter DNA methylation based on monocyte/macrophage might aid as diagnostic marker for clinical application or DNA methylation-related drug interventions may offer novel possibilities for atherosclerotic disease management.

Synthesis, bio-functionalization, and multifunctional activities of superparamagnetic-nanostructures have been extensively reviewed with a particular emphasis on their uses in a range of disease-specific biomarker detection and associated challenges.

• Colorectal neoplasms
• Heterogeneous methylation
• Interaction effect
• Peripheral blood

• Cervical Cancer
• Cervical Intraepithelial Neoplasia
• DNA Methylation

• CLL
• MS-HRM
• biomarker
• chronic lymphocytic leukemia
• heterogeneous methylation
• treatment

Background: Aberrant DNA methylation, especially tumor suppressor gene hypermethylation, is a well-recognized biomarker of initial tumorogenesis stages. FAT4 and SOX11 are putative tumor suppressor genes and can be down-regulated by hypermethylation in various cancers tissues. However, in peripheral blood leukocytes, the association between these two genes methylation status, as well as the effects of gene-environment interactions, and gastric cancer (GC) risk remain unclear.

Methods: A hospital-based case-control study including 375 cases and 394 controls was conducted. Peripheral blood leukocytes DNA methylation status were detected by methylation-sensitive high-resolution melting (MS-HRM) assay. Logistic regression was adopted to analyze the relationship of FAT4 and SOX11 methylation with GC susceptibility.

Results: Positive methylation (Pm) and total positive methylation (Tpm) of FAT4 were significantly increased the risk of GC (OR = 2.204, 95% CI: 1.168-4.159, P = 0.015; OR = 1.583, 95% CI: 1.031-2.430, P = 0.036, respectively). Compared with controls, cases exhibited higher SOX11 Pm frequencies with OR of 2.530 (95% CI: 1.289-4.969, P = 0.007). Nonetheless, no statistically significant association between SOX11 Tpm and GC risk was observed. Additionally, interactions between FAT4 Tpm and increased consumption of freshwater fish (≥1 times/week) displayed an antagonistic effect on GC (OR = 0.328, 95% CI: 0.142-0.762, P = 0.009), and high salt intake interacted with SOX11 Tpm also showed statistically significant (OR = 0.490, 95% CI: 0.242-0.995, P = 0.048).

Conclusions:FAT4 aberrant methylation in peripheral blood leukocytes and gene-environment interactions were associated with the risk of GC, while SOX11 was controversial and needed to be more investigated.

• DNA methylation
• gastric cancer
• gene-environment interaction.
• peripheral blood leukocytes

DNA methylation is closely associated with aberrant epigenetic changes. Previous studies have identified various genes associated with non-small cell lung cancer (NSCLC), but the precise combination responsible for its etiology is still debated. The aim of the present study was to select a new set of NSCLC-related genes using methylation-sensitive high-resolution melting. The promoter methylation status of six selected genes, consisting of protocadherin γ subfamily B, 6 (PCDHGB6), homeobox A9 (HOXA9), O₆-methylguanine-DNA methyltransferase (MGMT), microRNA (miR)-126, suppressor of cytokine signaling 3 (SOCS3) and Ras association domain family member 5, also termed NORE1A, was evaluated in 54 NSCLC patients. From these samples, genome-wide DNA was extracted and bisulfite conversion was performed along with fluorogenic quantitative polymerase chain reaction to detect methylation values of the six selected promoters. The present results revealed frequent methylation on PCDHGB6, HOXA9 and miR-126, which contrasted with infrequent methylation on MGMT. The results indicated no methylation on either SOCS3 or NORE1A. The sensitivity and specificity of the methylation assessment were 85.2 and 81.5%, respectively, and the analysis results were validated by pyrosequencing. Furthermore, minute comparison of the association between DNA methylation and clinical features was performed. Overall, these results may provide potential information for the development of better clinical diagnostics and more targeted and effective therapies for NSCLC.

• CpG
• diagnosis
• high-resolution melting
• lung cancer
• methylation

• DNA methylation
• EGR transcription factor
• MS-HRM
• RXRα
• Term pregnancy

• DNA methylation
• Heterogeneous methylation
• MS-HRM
• PCR bias
• Primer design

• RASSF1A
• lymph node status
• methylation
• methylation-specific polymerase chain reaction

• DNA methylation
• Lung adenocarcinoma
• Metastasis
• Non-small cell lung cancer
• miR-34 family
• microRNA

The association between the downregulation of genes and DNA methylation in their CpG islands has been extensively studied as a mechanism that favors carcinogenesis. The objective of this study was to analyze the methylation of a set of genes selected based on their microarray expression profiles during the process of hepatocarcinogenesis. Rats were euthanized at: 24 h, 7, 11, 16 and 30 days and 5, 9, 12 and 18 months post-treatment. We evaluated the methylation status in the CpG islands of four deregulated genes (Casp3, Cldn1, Pex11a and Nox4) using methylation-sensitive high-resolution melting technology for the samples obtained from different stages of hepatocarcinogenesis. We did not observe methylation in Casp3, Cldn1 or Pex11a. However, Nox4 exhibited altered methylation patterns, reaching a maximum of 10%, even during the early stages of hepatocarcinogenesis. We observed downregulation of mRNA and protein of Nox4 (97.5% and 40%, respectively) after the first carcinogenic stimulus relative to the untreated samples. Our results suggest that Nox4 downregulation is associated with DNA methylation of the CpG island in its promoter. We propose that methylation is a mechanism that can silence the expression of Nox4, which could contribute to the acquisition of neoplastic characteristics during hepatocarcinogenesis in rats.

Summary: Many genes downregulated by DNA methylation are tumor suppressor genes. In the present study, DNA methylation of Nox4 is observed, with implications for Nox4 as a potential therapeutic target for liver or other cancers.

• Casp3
• Chemical hepatocarcinogenesis
• Cldn1
• Downregulation
• Nox4, DNA methylation
• Pex11a

• cancer risk
• epigenetic
• genotoxicity
• occupational exposure
• toluidine

• Adult
• Aging/genetics
• Case-Control Studies
• Cross-Sectional Studies
• Cyclin-Dependent Kinase Inhibitor p16/genetics
• DNA Methylation
• DNA Modification Methylases/genetics
• DNA Repair Enzymes/genetics
• Female
• Glutathione S-Transferase pi/genetics
• Humans
• Middle Aged
• Nuclear Proteins/genetics
• Occupational Diseases/etiology
• Occupational Exposure/adverse effects
• Sweden
• Telomere
• Time Factors
• Tumor Suppressor Proteins/genetics
• Twist-Related Protein 1/genetics
• Young Adult

Preterm delivery and sub-optimal fetal growth are associated with each other and affect both mother and infant. Our aim was to determine (i) whether there are detectable differences in DNA methylation between early and late gestation and (ii) whether changes in DNA methylation from entry are associated with spontaneous preterm delivery with and without reduced fetal growth.

We conducted a case-control study nested within a large prospective cohort. Gene specific methylation was measured by Methyl-Profiler PCR Array in a Human Breast Cancer Signature Panel of 24 genes from maternal peripheral leukocytes genomic DNA at entry and 3^rd trimester (sampled at 16 and 30 weeks of gestation, respectively). Clonal bisulfite DNA sequencing was performed to confirm the changes in selected genes (CYP1B1, GADD45A and CXCL12). Multivariable analysis was used for data analysis.

There was significantly decrease in DNA methylation in 15 of 24 genes during the 3^rd trimester in cases of spontaneous preterm delivery (n=23) as compared to the controls (n=19) (p<0.05–p<0.01 for each gene). Similar results were observed by bisulfite sequencing for 3 genes. The change in DNA methylation between late and early gestation was significantly different in cases (overall decrease in methylation was −4.0 ± 1.5%) compared to the controls (overall increase in methylation was 12.6 ± 2.19%, p<0.0001). A graded pattern of DNA methylation was observed in 15 genes. Cases who delivered preterm with reduced fetal growth had the lowest level of methylation, cases delivering preterm without reduced fetal growth were next and term controls were highest in methylation (p for trend <0.05 to p<0.01 for each gene). Cases of preterm delivery also had significantly lower dietary choline intake.

These data suggest that epigenetic modification is associated with an increased risk of spontaneous preterm delivery, spontaneous preterm delivery with reduced fetal growth in particular.

• DNA methylation
• Free choline
• Reduced fetal growth
• Spontaneous preterm delivery
• Total choline
• Tumor related genes

• epigenetics
• psychosis
• mood disorder
• reln
• comt
• bdnf

• Bipolar Disorder/blood
• Bipolar Disorder/genetics
• DNA Methylation
• Humans
• Reelin Protein
• Saliva/metabolism
• Schizophrenia/blood
• Schizophrenia/genetics

• National Health and Medical Research Council
• Netherlands Institute for Advanced Study in the Humanities and Social Sciences (NL)
• Australian Research Council
• Australian Postgraduate Award

• biomarker
• environment
• exposure
• methylation

• Biomarker
• Formalin fixed paraffin embedded (FFPE)
• MS-HRM
• Methylation

• Carcinoma, Non-Small-Cell Lung/genetics
• DNA Methylation/genetics
• DNA, Neoplasm/analysis
• DNA, Neoplasm/metabolism
• Formaldehyde
• Genetic Techniques
• Humans
• Lung Neoplasms/genetics
• Paraffin Embedding
• Polymerase Chain Reaction
• Tissue Fixation

It has been shown in some articles that genetic and epigenetic abnormalities cannot only be found in tumor tissues but also in adjacent regions that appear histologically normal. This phenomenon is metaphorically called field cancerization or field defect. Field cancerization is regarded as clinically significant because it is assumed to be an important factor in local recurrence of cancer. As the field showing these molecular abnormalities may not be removed completely by surgery, these changes might lead to neoplasms and subsequent transformation to a tumor. We aimed to investigate the applicability of the methylation status of six tumor suppressor genes as biomarkers for detecting field cancerization in breast cancer.

The promoter methylation status of CCND2, DAPK1, GSTP1, HIN-1, MGMT and RASSF1A was determined by methylation-sensitive high-resolution melting (MS-HRM) analysis. MS-HRM methods for CCND2, MGMT and RASSF1A were developed in-house, primer sequences for DAPK1, GSTP1 and HIN-1 have already been published. Biopsy samples were taken from tumor, tumor-adjacent and tumor-distant tissue from 17 breast cancer patients. Normal breast tissues of four healthy women served as controls.

All MS-HRM methods proved to be very sensitive. LODs were in the range from 0.1 to 1.5 %, LOQs ranged from 0.3 to 5.3 %. A total of 94 %, 82 % and 65 % of the tumors showed methylation of RASSF1A, HIN-1 and MGMT promoters, respectively. The methylation status of these promoters was significantly lower in tumor-distant tissues than in tumor tissues. Tumor-adjacent tissues showed higher methylation status of RASSF1A, HIN-1 and MGMT promoters than tumor-distant tissues, indicating field cancerization. The methylation status of the HIN-1 promoter in tumor-adjacent tissues was found to correlate strongly with that in the corresponding tumors (r = 0.785, p < 0.001), but not with that in the corresponding tumor-distant tissues (r = 0.312, p = 0.239).

Among the gene promoters investigated, the methylation status of the HIN-1 promoter can be considered the best suitable biomarker for detecting field cancerization. Further investigation is needed to test whether it can be used for defining surgical margins in order to prevent future recurrence of breast cancer.

• DNA/analysis
• DNA/chemistry
• DNA/genetics
• DNA Methylation/genetics
• Equipment Design
• Humans
• Lab-On-A-Chip Devices
• MCF-7 Cells
• Microfluidic Analytical Techniques/instrumentation
• Microfluidic Analytical Techniques/methods
• Receptors, Retinoic Acid/genetics
• Sulfites/analysis
• Sulfites/chemistry

Despite similar clinical and pathological features, large numbers of breast cancer patients experience different outcomes of the disease. This, together with the fact that the incidence of breast cancer is growing worldwide, emphasizes an urgent need for identification of new biomarkers for early cancer detection and stratification of patients.

We used ultrahigh-resolution microarrays to compare genomewide methylation patterns of breast carcinomas (n = 20) and nonmalignant breast tissue (n = 5). Biomarker properties of a subset of discovered differentially methylated regions (DMRs) were validated using methylation-sensitive high-resolution melting (MS-HRM) in a case–control study on a panel of breast carcinomas (n = 275) and non-malignant controls (n = 74).

On the basis of microarray results, we selected 19 DMRs for large-scale screening of cases and controls. Analysis of the screening results showed that all DMRs tested displayed significant gains of methylation in the cancer tissue compared to the levels in control tissue. Interestingly, we observed two types of locus-specific methylation, with loci undergoing either predominantly full or heterogeneous methylation during carcinogenesis. Almost all tested DMRs (17 of 19) displayed low-level methylation in nonmalignant breast tissue, independently of locus-specific methylation patterns in cases.

Specific loci can undergo either heterogeneous or full methylation during carcinogenesis, and loci hypermethylated in cancer frequently show low-level methylation in nonmalignant tissue.

• Biosensing Techniques/methods
• Calorimetry/methods
• Cell Line, Tumor
• DNA Methylation
• DNA, Neoplasm/analysis
• DNA, Neoplasm/chemistry
• Gold/chemistry
• Humans
• Hydrophobic and Hydrophilic Interactions
• Metal Nanoparticles/chemistry
• Sulfites/chemistry

• Adaptor Proteins, Signal Transducing/chemistry
• Adaptor Proteins, Signal Transducing/isolation & purification
• Cyclin-Dependent Kinase Inhibitor p16/chemistry
• Cyclin-Dependent Kinase Inhibitor p16/genetics
• Cyclin-Dependent Kinase Inhibitor p16/isolation & purification
• DNA Fragmentation
• Denaturing Gradient Gel Electrophoresis
• Humans
• Limit of Detection
• Methylation
• Microfluidics/methods
• MutL Protein Homolog 1
• Nuclear Proteins/chemistry
• Nuclear Proteins/isolation & purification
• Promoter Regions, Genetic
• Sulfites/chemistry

• Base Sequence
• Cytosine/metabolism
• DNA/genetics
• DNA/metabolism
• DNA Methylation
• Electron Transport
• Optical Phenomena

The terms methylation quantification and qualification seem self-explanatory however, the results of experiments aiming to quantify or qualify locus specific methylation in clinical material are often difficult to interpret. There are three main reasons for difficulties in understanding methylation status measurement. First, the complexity of locus specific methylation patterns, which oscillate between unmethylated, fully methylated, and heterogeneously methylated. Second the interpretation of methylation-screening results can frequently be problematic due to limitations of the methods used. And finally the specifications of the clinical samples used in laboratory practice frequently hamper the methylation measurement. Thus, the process of quantification and qualification of methylation has to be discussed with consideration of the specific locus analyzed, the methodology used, and the clinical material source used in each specific experiment. The question of the clinical significance of determination of different methylation levels is even more complicated, with substantial evidence for correlation between qualitative methylation changes and clinical features of the disease and at the same time no data showing that different relative levels of methylation alter the disease outcome. The limitations of methylation quantification and qualification are discussed in this mini-review.

• MIP
• MSP
• cancer
• methylation
• qualification
• quantification

• Animals
• Cattle
• DNA Methylation
• DNA Modification Methylases/genetics
• DNA Modification Methylases/metabolism
• Endometrium/metabolism
• Epigenesis, Genetic
• Estrogen Receptor alpha/genetics
• Estrogen Receptor alpha/metabolism
• Estrous Cycle
• Female
• Gene Expression
• Polymerase Chain Reaction
• Pregnancy
• Pregnancy, Animal/genetics
• Pregnancy, Animal/metabolism
• RNA, Messenger/metabolism
• Regression Analysis
• Sequence Analysis, DNA/methods
• Transcription, Genetic
• Transition Temperature