|
| Status |
Public on Nov 01, 2011 |
| Title |
Patient 5 [CNV] |
| Sample type |
genomic |
| |
|
| Source name |
Lymphoblastoid cell line
|
| Organism |
Homo sapiens |
| Characteristics |
individual: Patient 5
cell line: Lymphoblastoid cell line
disease state: UPF3B patient
|
| Treatment protocol |
No treatment
|
| Growth protocol |
The Epstein-Barr virus (EBV) immortalized B- cell lines (B-LCL) used in this study were established from peripheral blood lymphocytes of patients and controls. Once established the LCL cell lines were cultured in RPMI 1640 (Sigma) supplemented with 10% FCS, 2mM L-Glutamine, 0.017mg/ml benzylpenicillin and grown at 37 0 C with 5% CO2
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The extraction was set up in a 1.5 ml Eppendorf screw cap tube (containing the pellet) with 400 µl of proteinase K buffer, 50 µl of 10% SDS and 50 µl of 10 mg/ml proteinase K (final concentration 1 mg/ml) (ROCHE). The tube was sealed off with paraffin to prevent leakage and spun on rotating wheel over night in 37oC incubator. DNA was separated from the lysate by adding 500 µl Phenol:Water:Chloroform mix and mixed well by rotating on wheel for 15 minutes at room Tm. To separate lysates into layers, the tube was spun at 12000 RPM for 5 minutes. The top aqueous phase, which contained DNA, was transferred into another 1.5 ml Eppendorf screw cap tube. To clean up more cell debris and protein contamination, another lot of 500 µl of Phenol:Water:Chloroform was added. The process was repeated until a cleaner top aqueous phase was collected in a different 1.5 ml Eppendorf screw cap tube. To remove RNA contamination, 2 µl of RNAse A (QIAGEN) was added to the DNA mixture and incubated at for 1 hour at 37oC. DNA was precipitated by adding ice cold absolute ethanol and incubated in -20oC freezer for 30 minutes, then spun at 12000 RPM for 10 minutes. All supernatant was removed and the DNA pellet was dried under vacuum. The dried DNA pellet was resuspended in 100-300 µl 1 x TE depending on the size of the original sample.
|
| Label |
Biotin
|
| Label protocol |
Performed by Australian Genomic Research Facility (AGRF)
|
| |
|
| Hybridization protocol |
Performed by Australian Genomic Research Facility (AGRF)
|
| Scan protocol |
Performed by Australian Genomic Research Facility (AGRF)
|
| Description |
LCL gDNA
|
| Data processing |
Raw data was originally processed by AGRF in Genome Bead Studio then exported to Partek Genomic Suite compatible files: allele intensity, genotype call and Ballele frequency files. Copy number was created from allele intentisity file against control 1 as reference model, then adjusted for GC content to reduce noise. CNV was detected in patients samples using Genomic Segmentation algorithm in Partek with the following settings: minimum markers 10, P value threshold < 0.001, signal to noise > 0.5, expected range = 0.3.
|
| |
|
| Submission date |
Feb 18, 2011 |
| Last update date |
Nov 01, 2011 |
| Contact name |
Jozef Gecz |
| Organization name |
SA Pathology
|
| Department |
Genetics Medicine
|
| Lab |
Neurogenetics
|
| Street address |
72 King William Rd
|
| City |
North Adelaide |
| State/province |
South Australia |
| ZIP/Postal code |
5006 |
| Country |
Australia |
| |
|
| Platform ID |
GPL13135 |
| Series (3) |
| GSE27125 |
Genome-wide consequences of compromised NMD and their relavence for variable clinical phenotype of patients with UPF3B mutations [mRNA profiling] |
| GSE27412 |
Genome-wide consequences of compromised NMD and their relavence for variable clinical phenotype of patients with UPF3B mutations [CNV] |
| GSE27433 |
Genome-wide consequences of compromised NMD and their relavence for variable clinical phenotype of patients with UPF3B mutations |
|
