|
| Status |
Public on Apr 28, 2012 |
| Title |
control sperm20 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
sperm, control
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: sperm
disease status: control
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The samples were first digested using proteinase K (20mg/ml) and lysis buffer mixture at 55°C overnight. A mixture of phenol, chloroform, and iso-amyl alcohol (in ratios of 25:24:1) was used to remove the proteins, followed by two times chloroform wash, precipitation of DNA using cold isopropanol, and a final wash using 70% ethanol. After the extraction, the DNA samples were suspended in 0.1M Tris-EDTA buffer and were kept in -80°C for storage prior to use. The purity and the concentrations of DNA were checked using a UV spectrophotometer (Nanodrop ND1000).
|
| Label |
Cy5
|
| Label protocol |
1 μg of purified enriched PCR products were fluorescently labelled with Cy3 (Roche) for the reference and with Cy5 (Roche) for the sample, as previously described (Schumacher, A., et al. (2006). Microarray-based DNA methylation profiling: technology and applications. Nucleic acids research 34, 528-542.) .
|
| |
|
| Channel 2 |
| Source name |
sperm, common reference pool
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: sperm
sample type: common reference pool
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The samples were first digested using proteinase K (20mg/ml) and lysis buffer mixture at 55°C overnight. A mixture of phenol, chloroform, and iso-amyl alcohol (in ratios of 25:24:1) was used to remove the proteins, followed by two times chloroform wash, precipitation of DNA using cold isopropanol, and a final wash using 70% ethanol. After the extraction, the DNA samples were suspended in 0.1M Tris-EDTA buffer and were kept in -80°C for storage prior to use. The purity and the concentrations of DNA were checked using a UV spectrophotometer (Nanodrop ND1000).
|
| Label |
Cy3
|
| Label protocol |
1 μg of purified enriched PCR products were fluorescently labelled with Cy3 (Roche) for the reference and with Cy5 (Roche) for the sample, as previously described (Schumacher, A., et al. (2006). Microarray-based DNA methylation profiling: technology and applications. Nucleic acids research 34, 528-542.) .
|
| |
|
| |
| Hybridization protocol |
The enriched PCR products were hybridized overnight at 48°C onto the 8.1k human CpG island microarray (Microarray Facility, University Health Network, Toronto) as previously described (Schumacher, A., et al. (2006). Microarray-based DNA methylation profiling: technology and applications. Nucleic acids research 34, 528-542.) .
|
| Scan protocol |
The arrays were scanned using the Genepix 4000B scanner and Genepix Pro 6.0 (Axon Instruments). The automated photomultiplier (PMT) function of the scanner was used to optimize signal intensity and channel balances.
|
| Description |
Sperm20 vs. common reference pool
|
| Data processing |
Linear Models for Microarray Data (LIMMA) package (Bioconductor) for R was used for background correction, normalization, and to extract the signal intensities. Briefly, normexp was used for background correction, print-tip loess was used for within-array normalization, and Gquantile was used for between-array normalization.
|
| |
|
| Submission date |
Apr 25, 2012 |
| Last update date |
Apr 28, 2012 |
| Contact name |
Art Petronis |
| Organization name |
Centre for Addiction and Mental Health
|
| Department |
Campbell Family Mental Health Research Institute
|
| Lab |
Krembil Family Epigenetics Laboratory
|
| Street address |
250 College St
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5T1R8 |
| Country |
Canada |
| |
|
| Platform ID |
GPL10342 |
| Series (1) |
| GSE37579 |
DNA modification study of major depressive disorder: beyond locus-by-locus comparisons. |
|
