|
| Status |
Public on Aug 21, 2007 |
| Title |
13902_methylatedDNA_peripheralBlood |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
normal peripheral blood
|
| Organism |
Homo sapiens |
| Characteristics |
Normal peripheral blood is from a healthy donor, female, 35 yrs old
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
standard phenol-chloroform extraction
|
| Label |
Cy3
|
| Label protocol |
using random primer direct labeling by Bioprimer CGH labeling kit from Invitrogen (Cat. No. 18095-011)
|
| |
|
| Channel 2 |
| Source name |
in vitro methylated DNA
|
| Organism |
Homo sapiens |
| Characteristics |
Normal peripheral blood is from a healthy donor, female, 35 yrs old, treated with SSSI methylase
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
standard phenol-chloroform extraction
|
| Label |
Cy5
|
| Label protocol |
using random primer direct labeling by Bioprimer CGH labeling kit from Invitrogen (Cat. No. 18095-011)
|
| |
|
| |
| Hybridization protocol |
we used Agilent microarray protocols for hybridization, blocking and washing (details can be found at www.agilent.com). Briefly,labeled samples were hybridized to arrays in the presence of human Cot-1 DNA for 40 hr at 65°C. The arrays were washed by Agilent Oligo aCGH wash buffer 1 for 5 mins, wash buffer 2 for 1 min, Acetonitrile for 1 min, and Stabilization and drying solution for 30s.
|
| Scan protocol |
we used Agilent scanner and feature extraction software for data process.
|
| Description |
The goal of the experiment – genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters
|
| Data processing |
normalization using LOWESS method.
The LogRatio was calculated based on:
If red channel signal is positive and significant vs background and greater than red background SD (R) and green channel signal is positive and significant vs background and greater than green background SD (G), then the ratio is R/G, and feature extraction uses the dye-normalized signals for both channels for the calculations.
If red channel signal is not positive and significant vs background or less than red background SD (r) and green channel signal is Positive and significant vs background and greater than green background SD (G), then ratio is r/G, and feature extraction uses the dye-normalized green signal and the dye-normalized red background SD signal (surrogate) for the calculations.
If red channel signal is not positive and significant vs background or less than red background SD (R), and green channel signal is not positive and significant vs background or less than green background SD (g), then ratio is r/g and feature extraction uses surrogates for both channels, and sets the log ratio=0, set the p-value=1.
If red channel signal is positive and significant vs background and greater then red background SD (R), and green channel signal is not positive and significant vs background or less than green background SD (g), then ratio is R/g, and feature extraction uses the dye-normalized red signal and the dye-normalized red signal and the dye-normalized green background SD signal (surrogate) for the calculations.
Gene selection procedure:
Probe sequences were downloaded from the Agilent website at www.agilent.com. Each probe was BLASTed against all sequences in the SmaI/XmaI database using BLAST v2.2.8 downloaded from NCBI (/data/bioinfo/SequenceTools/blast/). Probes with multiple BLAT hits were excluded from further study. Probes residing in SmaI/XmaI fragments were identified with the annotation for fragment length, status of CGI and repetitive sequences. The signal intensity for the probes within the SmaI/XmaI fragments was adjusted for background and analyzed for the ratio between Cy3 and Cy5 signals. All data analysis (sensitivity and reproducibility, correlation between methylation level and chromosome location, CGI, and repetitive sequences) were carried out in Excel (Microsoft). The resulting data sets are accessible in the supplementary database. We used the following criteria to select hypermethylated probes in PBL (Cy3) relative to fully methylated DNA (Cy5): signal intensity of Cy3>3 times background and ratio of Cy3/Cy5>=1.5. We performed bisulfite-pyrosequencing on 38 randomly selected genes showing higher signal intensity in PBL, and determined that these criteria most accurately identified hypermethylated loci.
|
| |
|
| Submission date |
Aug 17, 2007 |
| Last update date |
Aug 21, 2007 |
| Contact name |
Jiexin Zhang |
| E-mail(s) |
jiexinzhang@mdanderson.org
|
| Organization name |
UT MD Anderson Cancer Center
|
| Department |
Bioinformatics & Computational Biology
|
| Street address |
1515 Holcombe Blvd
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL4089 |
| Series (1) |
| GSE8810 |
methylatedDNA_peripheralBlood |
|
| Data table header descriptions |
| ID_REF |
|
| VALUE |
log ratio r/g |
| PValueLogRatio |
p-value obtained from log ratio |
| gSurrogateUsed |
surrogated used for green channel |
| rSurrogateUsed |
surrogated used for red channel |
| gProcessedSignal |
processed signal intensity for green channel |
| rProcessedSignal |
processed signal intensity for red channel |
| gDyeNormSignal |
dye-normalized signal for green channel |
| rDyeNormSignal |
dye-normalized signal for red channel |
| gNetSignal |
net signal intensity for green channel |
| rNetSignal |
net signal intensity for red channel |
