|
| Status |
Public on Jul 06, 2015 |
| Title |
CHLA-01-MED |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
original medulloblastoma tumor
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
cell type: Primary medulloblastoma
genotype: heterozygous BRCA2 6174delT
|
| Growth protocol |
None
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA from sorted nuclei was extracted using an amended protocol from QIAamp® DNA Micro Kit from Qiagen (Valencia, CA). Briefly each sorted sample was resuspended in 180ul buffer ATL and 20ul proteinase K then incubated for 3 hours at 56°C for complete lysis. Samples were bound and washed according to QIAamp® DNA Micro Kit instructions, eluted into 50ul of H20, then precipitated overnight with 5ul 3 M sodium acetate and 180 ul 100% EtOH. Each sample was then centrifuged for 30 minutes at 20,000 x g, washed in 1 ml of 70% EtOH for 30 minutes at 20,000 x g. The samples were carefully decanted and the DNA pellet was dried by speed vacuum then resuspension in a small volume (e.g. 10-50ul) of H20 for final concentrations suitable for accurate quantification.
|
| Label |
Cy5
|
| Label protocol |
Extracted fresh frozen sourced genomic DNA was amplified using the phi29 based Illustra GenomiPhi V2 Amplification kit from GE Healthcare Bio-sciences Corp (Piscataway,NJ) according to our published protocols18. A 100 ng aliquot of pooled 46,XX DNA (Promega, Madison, WI) was amplified with the matching amplification protocol to generate a suitable reference for each aCGH experiment using amplified DNA template. In all cases the quality of the amplification product was assessed by gel electrophoresis. Fresh frozen phi29 amplified and FFPE non-amplified DNAs were treated with DNAse 1 prior to Klenow based labeling. High molecular weight phi29 templates were digested for 30 minutes while the smaller fragmented FFPE samples were digested for only 1 minute. In each case 1ul of 10x DNase 1 reaction buffer and 2ul of DNase 1 dilution buffer were added to 7ul of DNA sample and incubated at room temperature then transferred to 70°C for 30 minutes to deactivate DNase 1. In contrast the amplified FFPE sourced DNAs do not require DNase 1 treatment prior to Klenow-based labeling. Sample and reference templates were then labeled with Cy-5 dUTP and Cy-3 dUTP respectively using a BioPrime labeling kit (Invitrogen, Carlsbad, CA) according to our published protocols
|
| |
|
| Channel 2 |
| Source name |
Reference pooled 46XX
|
| Organism |
Homo sapiens |
| Characteristics |
reference type: Normal female genome
|
| Growth protocol |
None
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA from sorted nuclei was extracted using an amended protocol from QIAamp® DNA Micro Kit from Qiagen (Valencia, CA). Briefly each sorted sample was resuspended in 180ul buffer ATL and 20ul proteinase K then incubated for 3 hours at 56°C for complete lysis. Samples were bound and washed according to QIAamp® DNA Micro Kit instructions, eluted into 50ul of H20, then precipitated overnight with 5ul 3 M sodium acetate and 180 ul 100% EtOH. Each sample was then centrifuged for 30 minutes at 20,000 x g, washed in 1 ml of 70% EtOH for 30 minutes at 20,000 x g. The samples were carefully decanted and the DNA pellet was dried by speed vacuum then resuspension in a small volume (e.g. 10-50ul) of H20 for final concentrations suitable for accurate quantification.
|
| Label |
Cy3
|
| Label protocol |
Extracted fresh frozen sourced genomic DNA was amplified using the phi29 based Illustra GenomiPhi V2 Amplification kit from GE Healthcare Bio-sciences Corp (Piscataway,NJ) according to our published protocols18. A 100 ng aliquot of pooled 46,XX DNA (Promega, Madison, WI) was amplified with the matching amplification protocol to generate a suitable reference for each aCGH experiment using amplified DNA template. In all cases the quality of the amplification product was assessed by gel electrophoresis. Fresh frozen phi29 amplified and FFPE non-amplified DNAs were treated with DNAse 1 prior to Klenow based labeling. High molecular weight phi29 templates were digested for 30 minutes while the smaller fragmented FFPE samples were digested for only 1 minute. In each case 1ul of 10x DNase 1 reaction buffer and 2ul of DNase 1 dilution buffer were added to 7ul of DNA sample and incubated at room temperature then transferred to 70°C for 30 minutes to deactivate DNase 1. In contrast the amplified FFPE sourced DNAs do not require DNase 1 treatment prior to Klenow-based labeling. Sample and reference templates were then labeled with Cy-5 dUTP and Cy-3 dUTP respectively using a BioPrime labeling kit (Invitrogen, Carlsbad, CA) according to our published protocols
|
| |
|
| |
| Hybridization protocol |
All labeling reactions were assessed using a Nanodrop assay (Nanodrop, Wilmington, DE) prior to mixing and hybridization to 400k CGH arrays (Agilent Technologies, Santa Clara, CA) for 40 hours in a rotating 65°C oven. All hybridizations were done with a pooled commercial 46,XX reference (Promega, Madison WI GSM540609).
|
| Scan protocol |
All microarray slides were scanned using an Agilent 2565C DNA scanner and the images were analyzed with Agilent Feature Extraction version 10.7 using default settings.
|
| Data processing |
The aCGH data was assessed with a series of QC metrics,dye normalized and centralized, then analyzed using an aberration detection algorithm (ADM2) which is included in Agilent Genomic Workbench 6.5 software. The latter identifies all aberrant intervals in a given sample with consistently high or low log ratios based on the statistical score derived from the average normalized log ratios of all probes in the genomic interval multiplied by the square root of the number of these probes. This score represents the deviation of the average of the normalized log ratios from its expected value of zero and is proportional to the height h (absolute average )
|
| |
|
| Submission date |
Jun 18, 2015 |
| Last update date |
Jul 06, 2015 |
| Contact name |
Michael Thomas Barrett |
| E-mail(s) |
[email protected]
|
| Phone |
480-301-6736
|
| Organization name |
Mayo Clinic Arizona
|
| Department |
Molecular Pharmacology and Experimental Therapeutics
|
| Street address |
13400 East Shea Boulevard
|
| City |
Scottsdale |
| State/province |
AZ |
| ZIP/Postal code |
85259 |
| Country |
USA |
| |
|
| Platform ID |
GPL19387 |
| Series (1) |
| GSE70003 |
Disseminated medulloblastoma in a child with germline BRCA2 6174delT mutation and without Fanconi anemia |
|
