|
| Status |
Public on Aug 29, 2024 |
| Title |
Patient 3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Blood from cancer patient
|
| Organism |
Homo sapiens |
| Characteristics |
gender: Female
cell type: cancer
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA extracted and purified by different methods (extraction on Qiagen columns and purification on Amicon columns, extraction with DNA extractor robots [STARLET, QIACube]). The sample is measured (BMO.NANO.APP or BMO.QB.APP) and diluted in Nuclease Free water to obtain the desired final concentration in an amber Eppendorf: generally 500-400ng or 200ng depending on the protocol used in a volume of 20.2µL for 180K Agilent method chips and in a volume of 10µL for 60K ENZO method chips. At the same time, a “control” sample is prepared for each patient using the same process as the “patient” sample (same concentration, same sex).
|
| Label |
Cy5
|
| Label protocol |
Spin the samples in a centrifuge for 1 minute at 6,000 × g to drive the contents off the walls and lid. Add Random Primers. Set the thermal cycler to run the program. Transfer the plate or tubes into the thermal cycler, and then start the program. Remove the samples from the thermal cycler. Spin the samples in a centrifuge for 1 minute at 6,000 × g to drive the contents off the walls and lid. Mix the components on ice in the order indicated to prepare one cyanine-3 and one cyanine-5 Labeling Master Mix. Add 19 μL (or 21 μL) of Labeling Master Mix to each reaction tube containing the gDNA to make a total volume of 50 μL. Mix well by gently pipetting up and down. Set the thermal cycler to run the program. At the end of the thermal cycler program, remove the samples from the thermal cycler. Spin them briefly in a centrifuge to drive the contents off the walls and lid. Put samples on ice.
|
| |
|
| Channel 2 |
| Source name |
Agilent control
|
| Organism |
Homo sapiens |
| Characteristics |
gender: Female
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA extracted and purified by different methods (extraction on Qiagen columns and purification on Amicon columns, extraction with DNA extractor robots [STARLET, QIACube]). The sample is measured (BMO.NANO.APP or BMO.QB.APP) and diluted in Nuclease Free water to obtain the desired final concentration in an amber Eppendorf: generally 500-400ng or 200ng depending on the protocol used in a volume of 20.2µL for 180K Agilent method chips and in a volume of 10µL for 60K ENZO method chips. At the same time, a “control” sample is prepared for each patient using the same process as the “patient” sample (same concentration, same sex).
|
| Label |
Cy3
|
| Label protocol |
Spin the samples in a centrifuge for 1 minute at 6,000 × g to drive the contents off the walls and lid. Add Random Primers. Set the thermal cycler to run the program. Transfer the plate or tubes into the thermal cycler, and then start the program. Remove the samples from the thermal cycler. Spin the samples in a centrifuge for 1 minute at 6,000 × g to drive the contents off the walls and lid. Mix the components on ice in the order indicated to prepare one cyanine-3 and one cyanine-5 Labeling Master Mix. Add 19 μL (or 21 μL) of Labeling Master Mix to each reaction tube containing the gDNA to make a total volume of 50 μL. Mix well by gently pipetting up and down. Set the thermal cycler to run the program. At the end of the thermal cycler program, remove the samples from the thermal cycler. Spin them briefly in a centrifuge to drive the contents off the walls and lid. Put samples on ice.
|
| |
|
| |
| Hybridization protocol |
Prepare the 10× Blocking Agent. Prepare labeled gDNA for hybridization. Prepare the hybridization assembly. Hybridize.
|
| Scan protocol |
Put assembled slide holders into the scanner cassette. Select Protocol AgilentG3_CGH for G3 microarrays. Select Protocol AgilentHD_CGH for HD microarrays. Verify that the Scanner status in the main window says Scanner Ready. Click Start Scan.
|
| Description |
ch1: patient, ch2: control
Agilent, ISCA CGH 60K (AMADID 031746)
|
| Data processing |
After scanning is completed, extract features and analyze. Feature extraction is the process by which data is extracted from the scanned microarray image (.tif) and translated into log ratios, allowing researchers to identify aberrations in their samples. Agilent provides Feature Extraction software as a standalone program and as an integral part of CytoGenomics software (Windows version only).
Set up Analysis Workflow in the CytoGenomics program to analyze your data. You can use the Agilent Euro Female or Agilent Euro Male files as the Reference. The dbSNP141 files contain all of the active SNP probes of the Euro files, plus 50,000 new probes.
|
| |
|
| Submission date |
Jul 04, 2024 |
| Last update date |
Aug 29, 2024 |
| Contact name |
Maria Valeria Freire |
| Organization name |
Uliege
|
| Department |
Human Genetics
|
| Street address |
Av. Hippocrate 1/11
|
| City |
Liège |
| ZIP/Postal code |
4000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL21461 |
| Series (1) |
| GSE271498 |
Genetic of multiple primary cancers |
|
