|
| Status |
Public on Feb 26, 2019 |
| Title |
TGCT#3-A1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Testicular germ cell cancer
|
| Organism |
Homo sapiens |
| Characteristics |
origin: Recurrant
site: Primary
ploidy: 3.0N
|
| Growth protocol |
Formalin fixed paraffin embedded tumor samples were obtained from and reviewed by the Mayo Clinic Department of Anatomic Pathology
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Excess paraffin is removed with a scalpel from individual 40-60um scrolls which are then washed with 1ml Xylene for 5 minutes. The samples are filtered through a 35um mesh and resuspended in a final concentration of 10ug/ml DAPI prior to flow sorting with an Influx cytometer (Becton-Dickinson, San Jose, CA) with ultraviolet excitation and DAPI emission collected at >450 nm. DNA content and cell cycle were analyzed using the software program MultiCycle (Phoenix Flow Systems, San Diego, CA). DNAs were extracted using Qiagen micro kits (Qiagen Valencia, CA).
|
| Label |
Cy5
|
| Label protocol |
DNAs from FFPE-derived DNA samples were digested for 1 minute with DNAse1. In each case 1 ul of 10x DNase 1 reaction buffer and 2 μl of DNase 1 dilution buffer were added to 7 μl of DNA sample and incubated at room temperature then transferred to 70°C for 30 minutes to deactivate DNase 1. 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 [22]. All labeling reactions were assessed using a Nanodrop assay (Nanodrop, Wilmington, DE) prior to mixing and hybridization to CGH arrays (Agilent Technologies, Santa Clara, CA).
|
| |
|
| Channel 2 |
| Source name |
Reference pooled 46XX
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Normal female genome
|
| Growth protocol |
Formalin fixed paraffin embedded tumor samples were obtained from and reviewed by the Mayo Clinic Department of Anatomic Pathology
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Excess paraffin is removed with a scalpel from individual 40-60um scrolls which are then washed with 1ml Xylene for 5 minutes. The samples are filtered through a 35um mesh and resuspended in a final concentration of 10ug/ml DAPI prior to flow sorting with an Influx cytometer (Becton-Dickinson, San Jose, CA) with ultraviolet excitation and DAPI emission collected at >450 nm. DNA content and cell cycle were analyzed using the software program MultiCycle (Phoenix Flow Systems, San Diego, CA). DNAs were extracted using Qiagen micro kits (Qiagen Valencia, CA).
|
| Label |
Cy3
|
| Label protocol |
DNAs from FFPE-derived DNA samples were digested for 1 minute with DNAse1. In each case 1 ul of 10x DNase 1 reaction buffer and 2 μl of DNase 1 dilution buffer were added to 7 μl of DNA sample and incubated at room temperature then transferred to 70°C for 30 minutes to deactivate DNase 1. 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 [22]. All labeling reactions were assessed using a Nanodrop assay (Nanodrop, Wilmington, DE) prior to mixing and hybridization to CGH arrays (Agilent Technologies, Santa Clara, CA).
|
| |
|
| |
| Hybridization protocol |
Labeled DNA was hybridized without ozone scavenger and chips washed according to manufacture's protocol for 40 hours in a rotating 65°C oven.
|
| Scan protocol |
All microarray slides were scanned using an Agilent 2565C DNA scanner and the images were analyzed with Agilent Feature Extraction version 11.0 using default settings.
|
| Data processing |
Data was extracted from the TIFF files using Agilent FE 10. The aCGH data was assessed with a series of QC metrics then analyzed using an aberration detection algorithm (ADM2)[23]. 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 log ratio) of the genomic interval, and to the square root of the number of probes in the interval.
|
| |
|
| Submission date |
Dec 06, 2018 |
| Last update date |
Feb 28, 2019 |
| Contact name |
Michael Thomas Barrett |
| E-mail(s) |
barrett.michael@mayo.edu
|
| 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) |
| GSE123464 |
Clonal Analyses of Refractory Testicular Germ Cell Tumors |
|
