|
| Status |
Public on Oct 01, 2020 |
| Title |
T341_Xenograft_Generation_3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Xenotransplant
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Xenotransplant
gender: Female
|
| Growth protocol |
patient specimen were: 1) flash frozen on dry ice after surgical removal or 2) mechanically cut, seeded on agar coated flasks (0.85%) and allowed to form spheroids for up to 2 weeks at 37°C under 5% CO2 and atmospheric oxygen in DMEM medium, 10% FBS, 2mM L-Glutamine, 0.4mM NEAA and 100U/ml Pen-Strep (all from Lonza). Spheroids (generation 0) with a diameter of 300-1000 µm were then implanted in the brain of immunodeficient mice (NOD/Scid, Nude or NSG; 6 spheroids per mice). Cell lines were derived from xenotransplanted mice by papain-based enzymatic digestion of PDOX tissue and cultured in serum-free medium based on Neurobasal® base medium (Life Technologies) supplemented with 1 x B27 (Life Technologies) 2 mM L-Glutamine, 30 U/ml Pen-Step, 1 U/ml Heparin (Sigma), 20 ng/ml bFGF (Miltenyi, 130-093-841) and 20 ng/ml EGF (Provitro, 1325950500).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from patient tissue, spheroid cultures or cell lines using the AllPrep DNA/RNA Mini Kit® (Qiagen) following manufacturer’s instructions for “Simultaneous purification of genomic DNA and total RNA from animal tissues”.
|
| Label |
Cy5
|
| Label protocol |
For Agilent-021529 platform 2µg, for Agilent-021850 and Agilent-028081 platform 1µg, for Agilent-030587 platform 500ng and for Agilent-021924 platform 250ng of genomic DNA were digested with RSA1 and Alu1 (Agilent) to generate 200-500bp large fragments. The BioPrime aCGH Genomic labeling Kit (Life Technologies) and Cy3 and Cy5 dyes (GE Healthcare) were used following standard protocols for Agilent aCGH (CGH enzymatic protocol v6.2; Ref # G4410-90010).
|
| |
|
| Channel 2 |
| Source name |
Reference DNA
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Reference DNA
gender: Female
catalog no: G1521
|
| Growth protocol |
patient specimen were: 1) flash frozen on dry ice after surgical removal or 2) mechanically cut, seeded on agar coated flasks (0.85%) and allowed to form spheroids for up to 2 weeks at 37°C under 5% CO2 and atmospheric oxygen in DMEM medium, 10% FBS, 2mM L-Glutamine, 0.4mM NEAA and 100U/ml Pen-Strep (all from Lonza). Spheroids (generation 0) with a diameter of 300-1000 µm were then implanted in the brain of immunodeficient mice (NOD/Scid, Nude or NSG; 6 spheroids per mice). Cell lines were derived from xenotransplanted mice by papain-based enzymatic digestion of PDOX tissue and cultured in serum-free medium based on Neurobasal® base medium (Life Technologies) supplemented with 1 x B27 (Life Technologies) 2 mM L-Glutamine, 30 U/ml Pen-Step, 1 U/ml Heparin (Sigma), 20 ng/ml bFGF (Miltenyi, 130-093-841) and 20 ng/ml EGF (Provitro, 1325950500).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from patient tissue, spheroid cultures or cell lines using the AllPrep DNA/RNA Mini Kit® (Qiagen) following manufacturer’s instructions for “Simultaneous purification of genomic DNA and total RNA from animal tissues”.
|
| Label |
Cy3
|
| Label protocol |
For Agilent-021529 platform 2µg, for Agilent-021850 and Agilent-028081 platform 1µg, for Agilent-030587 platform 500ng and for Agilent-021924 platform 250ng of genomic DNA were digested with RSA1 and Alu1 (Agilent) to generate 200-500bp large fragments. The BioPrime aCGH Genomic labeling Kit (Life Technologies) and Cy3 and Cy5 dyes (GE Healthcare) were used following standard protocols for Agilent aCGH (CGH enzymatic protocol v6.2; Ref # G4410-90010).
|
| |
|
| |
| Hybridization protocol |
Similarly treated amounts of reference; Female or Male gDNA pool (Promega) were processed as sample genomic DNAs and hybridized with the Agilent Oligo aCGH Hybridization Kit to the corresponding platform array at 65°C for 24h or 48h respectively.
|
| Scan protocol |
Microarray slides were scanned using an Agilent 2565C DNA scanner
|
| Description |
T341_X3
|
| Data processing |
The tif images were analyzed with Agilent Feature Extraction version 12.5, using default settings
normalised log ratio Cy5/Cy3 representing test/reference; transformed logratio matrix with 0 - unaltered, 1 - gain, -1 - loss, 2 - amplification, -2 - deletion
|
| |
|
| Submission date |
Sep 24, 2019 |
| Last update date |
Oct 01, 2020 |
| Contact name |
Ann-Christin Hau |
| E-mail(s) |
ann-christin.hau@lns.etat.lu
|
| Organization name |
Laboratoire National de sante
|
| Department |
National Center of Pathology
|
| Street address |
1, Rue Louis Rech
|
| City |
Luxembourg |
| ZIP/Postal code |
3555 |
| Country |
Luxembourg |
| |
|
| Platform ID |
GPL16237 |
| Series (1) |
| GSE137959 |
Copy-Number abberation profiles of glioma patient tissue samples and corresponding xenografted tumor samples as well as cell lines derived there-of |
|
