|
| Status |
Public on Sep 03, 2012 |
| Title |
5798 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
FTD-XY
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
cell type: white blood cells
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA from patients as well as from healthy controls was isolated from peripheral blood using the Wizard Genomic DNA purification Kit (Promega, Madison, WI) and using manufacturer’s protocol.
|
| Label |
Cy5
|
| Label protocol |
500 ng genomic DNA was digested with AluI/RsaI restriction enzyme mix (Promega, Madison, WI). Fragmented DNA was labeled by direct enzymatic incorporation of fluorescent tags. Genomic DNA was labeled with Cy5-dUTP using the Agilent Genomic DNA Labeling kit PLUS (Agilent Technologies). Briefly, 50 ul reaction mix was incubated with Exo-Klenow Fragment at 37°C for 2 hrs. Unincorporated nucleotides were removed on Microcon YM-30 filters (Millipore, Billerica, MA) according to the manufacturer protocol.
|
| |
|
| Channel 2 |
| Source name |
Co1-XY
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
cell type: white blood cells
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA from patients as well as from healthy controls was isolated from peripheral blood using the Wizard Genomic DNA purification Kit (Promega, Madison, WI) and using manufacturer’s protocol.
|
| Label |
Cy3
|
| Label protocol |
500 ng genomic DNA was digested with AluI/RsaI restriction enzyme mix (Promega, Madison, WI). Fragmented DNA was labeled by direct enzymatic incorporation of fluorescent tags. Genomic DNA was labeled with Cy3-dUTP using the Agilent Genomic DNA Labeling kit PLUS(Agilent Technologies). Briefly, 50 ul reaction mix was incubated with Exo-Klenow Fragment at 37°C for 2 hrs. Unincorporated nucleotides were removed on Microcon YM-30 filters (Millipore, Billerica, MA) according to the manufacturer protocol.
|
| |
|
| |
| Hybridization protocol |
Dye incorporations of post-labeling products were measured by ND-1000 spectrophotometer (NanoDrop Technologies). Fluorescent-labeled reference and disease DNAs were mixed with 50 ug of human Cot-1 DNA (Invitrogen Life Technologies) and were hybridized to Human custom Ag-VIBxp1122v2-4X44 CGH Microarray (Agilent Technologies). Slides were hybridized using a Hybridization gasket slide (Agilent Technologies), placed in rotisserie (20 RPM rotation speed) in the hybridization oven at 65° C for 24 hrs (Agilent Technologies). After hybridization the slides were washed with Agilent washing solutions (including Stabilization and Drying solutions), according to Agilent Oligonucleotide array-based CGH for genomic DNA analysis protocol (version 6.0).
|
| Scan protocol |
After washing, the slides were scanned at 5 µm resolution by using a DNA microarray scanner (Agilent Technologies). Scan settings: Scan region was set to Scan Area (61 × 21.6 mm), Green PMT was set to 100%, Red PMT was set to 100%. Images were processed with the Feature Extraction Software version 9.5.3.1 (Agilent Technologies).
|
| Description |
Patients with X-linked intellectual disability (XLID)
|
| Data processing |
Prior to normalization, data from control and empty spots was removed. Due to the large number of probes on the X chromosome that are duplicated, the Loess based normalization as Prior to normalization, data from control and empty spots was removed. Due to the large number of probes on the X chromosome that are duplicated, the Loess based normalization as performed within the Agilent Feature extraction software (version 9.5.3.1) fails. Therefore, we fit the Loess regression line through a subset of the data, i.e., those probes that are not located in the region that is expected to be duplicated/deleted. We base ourselves on the uncorrected intensities obtained with the Agilent Feature extraction software (version 9.5.3.1) and we base our Loess fit on the subset of clones that map outside of the region 52.8 and 54.0Mb on the X chromosome. We omit data from unmapped clones and from spots that were not above background in both channels. The presented values are log-(Cy5/Cy3)-ratios. In case of multiple probes for the same Agilent ID, log-ratios were averaged.
|
| |
|
| Submission date |
Oct 13, 2011 |
| Last update date |
Sep 03, 2012 |
| Contact name |
Rekin's Janky |
| E-mail(s) |
[email protected]
|
| Organization name |
VIB
|
| Department |
Nucleomics Core
|
| Street address |
Herestraat 49 Box 816
|
| City |
Leuven |
| ZIP/Postal code |
B-3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL14729 |
| Series (1) |
| GSE32945 |
Mapping of the HUWE1 duplication at Xp11.22 in eight unrelated XLID patients |
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