|
| Status |
Public on Jul 06, 2016 |
| Title |
PO_086 stress |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
PO_086_meDIP
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
protocol: high levels of prenatal stress
chip antibody: anti-5-methylcytidine (33D3) antibody (Eurogentec)
tissue: cord blood
|
| Growth protocol |
Data were obtained from a cohort of mothers and their infants (n = 180) recruited during the third trimester of pregnancy in the Rhine-Neckar Region of Germany. A structured interview and questionnaires were used for risk factor assessment (Supplementary Table S1a). A composite score was calculated to take three different dimensions of stress into account: a) maternal psychopathology (primarily depressive and anxiety symptoms); b) perceived stress; and c) socioeconomic and psychosocial stress (Supplementary Table S1c). Stressful prenatal adverse conditions were also considered in order to define 10 infants with extremely high and 10 infants with extremely low levels of prenatal ELS respectively.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cord blood was collected immediately after birth and drawn into ethylenediaminetetraacetic (EDTA) coated tubes. Peripheral blood mononuclear cells (PBMCs) were isolated through centrifugation with Ficoll-Paque (GE Healthcare; Munich; Germany), and CD34+ cells were extracted from PBMCs using the Dynal CD34 Progenitor Cell Selection System (life technologies; Darmstadt; Germany).
|
| Label |
Cy5
|
| Label protocol |
After the sonication step, methylated DNA was immunoprecipitated with 10 μg of anti-5methylCytosine antibody (Eurogentec, Fremont, CA, USA) that is specific to DNA methylation, does not recognize hydroxymethylated DNA, and reveals changes in 5-methyl-cytosine exclusively. The DNA antibody complex was immunoprecipitated with protein G, and the methylated DNA was re-suspended in 0.25 ml of digestion buffer (50 mM TRisHCl pH = 8; 10 mM EDTA; 0.5 % SDS) and treated with 40 μg of proteinase K overnight at 55°C. The input and bound fractions were phenol/chloroform-extracted and ethanol-precipitated The input and bound fractions were amplified using the Whole Genome Amplification Kit (Sigma-Aldrich; St. Luis; MO; USA) and then labeled for microarray hybridization with Cy3-dUTP and Cy5-dUTP, respectively, using the CGH Enzymatic Labeling Kit (Agilent Technologies; Mississauga; ON; Canada) in accordance with the manufacturer´s instructions.
|
| |
|
| Channel 2 |
| Source name |
PO_086_input_DNA
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
protocol: high levels of prenatal stress
chip antibody: none
tissue: cord blood
|
| Growth protocol |
Data were obtained from a cohort of mothers and their infants (n = 180) recruited during the third trimester of pregnancy in the Rhine-Neckar Region of Germany. A structured interview and questionnaires were used for risk factor assessment (Supplementary Table S1a). A composite score was calculated to take three different dimensions of stress into account: a) maternal psychopathology (primarily depressive and anxiety symptoms); b) perceived stress; and c) socioeconomic and psychosocial stress (Supplementary Table S1c). Stressful prenatal adverse conditions were also considered in order to define 10 infants with extremely high and 10 infants with extremely low levels of prenatal ELS respectively.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cord blood was collected immediately after birth and drawn into ethylenediaminetetraacetic (EDTA) coated tubes. Peripheral blood mononuclear cells (PBMCs) were isolated through centrifugation with Ficoll-Paque (GE Healthcare; Munich; Germany), and CD34+ cells were extracted from PBMCs using the Dynal CD34 Progenitor Cell Selection System (life technologies; Darmstadt; Germany).
|
| Label |
Cy3
|
| Label protocol |
After the sonication step, methylated DNA was immunoprecipitated with 10 μg of anti-5methylCytosine antibody (Eurogentec, Fremont, CA, USA) that is specific to DNA methylation, does not recognize hydroxymethylated DNA, and reveals changes in 5-methyl-cytosine exclusively. The DNA antibody complex was immunoprecipitated with protein G, and the methylated DNA was re-suspended in 0.25 ml of digestion buffer (50 mM TRisHCl pH = 8; 10 mM EDTA; 0.5 % SDS) and treated with 40 μg of proteinase K overnight at 55°C. The input and bound fractions were phenol/chloroform-extracted and ethanol-precipitated The input and bound fractions were amplified using the Whole Genome Amplification Kit (Sigma-Aldrich; St. Luis; MO; USA) and then labeled for microarray hybridization with Cy3-dUTP and Cy5-dUTP, respectively, using the CGH Enzymatic Labeling Kit (Agilent Technologies; Mississauga; ON; Canada) in accordance with the manufacturer´s instructions.
|
| |
|
| |
| Hybridization protocol |
All the steps of hybridization, washing, scanning and feature extraction were performed following the Agilent protocols for chip-on-chip analysis (MeDIP).
|
| Scan protocol |
Scanning was performed using the Agilent Technologies Scanner G2505B. Probe intensities were extracted from scanned microarray images using Agilent's Feature Extraction 10.7.3.1 Image Analysis Software
|
| Data processing |
The extracted intensities were then analyzed using the R software environment for statistical computing (R Development Core Team, 2007) (http://www.r-project.org/). Log-ratios of the bound (Cy5) and input (Cy3) microarray channel intensities were computed for each microarray. Microarray quality was assessed using plots generated by the feature extraction software in addition to correlation matrix visualizations. Microarrays were normalized using quantile-normalization 8. Estimates of DNA methylation levels based on microarray probe intensities were obtained using a Bayesian deconvolution algorithm. Differential methylation between groups was determined in two stages. The first stage used linear models implemented in the ‘limma’ package 10 of Bioconductor 11 to compute a modified t-statistic at the individual probe level. As in typical microarray studies, the number of sample profiles is small so there is little information per probe from which to estimate probe variance. Hence, the modified t-statistic for each probe makes use of variance estimates derived from the other probes on the microarray. Correlation between technical replicates was modeled as a random effect using the “block” variable. An individual probe was called differentially methylated if the significance of its t-statistic was at most 0.05 (uncorrected for multiple testing) and the associated difference of log-normalized means between the groups was at least 0.5. In the second stage, differential statistics per promoter were derived from the t-statistics of the probes within the promoter. In particular, the Wilcoxon rank-sum test was used to identify enrichment for probes within the promoter having large positive or large negative t-statistics. The resulting promoter p-values were adjusted for multiple testing by calculating false discovery rates using the Benjamini-Hochberg algorithm. A promoter was called differentially methylated if its false discovery rate was at most 0.2 and it contained at least one probe called differentially methylated, as defined above.
|
| |
|
| Submission date |
Jul 05, 2016 |
| Last update date |
Jul 06, 2016 |
| Contact name |
Moshe Szyf |
| E-mail(s) |
moshe.szyf@mcgill.ca
|
| Organization name |
McGill University
|
| Department |
Pharmacology
|
| Street address |
McIntyre Medical Building, 3655 Promenade Sir William Osler, Room 1309
|
| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H3G 1Y6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL22110 |
| Series (2) |
| GSE84018 |
DNA methylation in CD34 positive cells derived from cord blood at birth following prenatal stress |
| GSE84028 |
Ankyrin-3 as a Molecular Marker of Early Life Stress and Vulnerability to Psychiatric Disorders |
|
