|
| Status |
Public on Oct 05, 2011 |
| Title |
TIL_deletion_characterization |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
wholeblood leukocytes
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: wholeblood leukocytes
genotype/variation: TIL_deletion
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
High-molecular-weight DNAs from patients and healthy donors were prepared by standard proteinase K digestion followed by phenol-chloroform extraction from wholeblood leukocytes.
The quantity and quality of the DNA samples were assessed with Nanodrop technology (Coleman Technologies, Orlando, FL) and verified by electrophoresis through agarose gels and staining with ethidium bromide.
|
| Label |
Cy5
|
| Label protocol |
Genomic DNA from each sample was double digested with AluI and RsaI (Promega, Madison, WI) for 2 hours at 37°C. The digested DNAs were labeled by random priming with the Agilent Genomic DNA Labeling Kit Plus (Agilent technologies) for 2 hours at 37°C, according to the manufacturer’s instructions (Protocol v6.3, October 2010, Agilent technologies). Patients’ DNA and pooled normal control DNAs (reference) were labeled with Cy5-dUTP and Cy3-dUTP, respectively.
|
| |
|
| Channel 2 |
| Source name |
Pool of six normal control DNAs from six healthy donors
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: wholeblood leukocytes
reference sample: Pool of six normal control DNAs from six healthy donors
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
High-molecular-weight DNAs from patients and healthy donors were prepared by standard proteinase K digestion followed by phenol-chloroform extraction from wholeblood leukocytes.
The quantity and quality of the DNA samples were assessed with Nanodrop technology (Coleman Technologies, Orlando, FL) and verified by electrophoresis through agarose gels and staining with ethidium bromide.
|
| Label |
Cy3
|
| Label protocol |
Genomic DNA from each sample was double digested with AluI and RsaI (Promega, Madison, WI) for 2 hours at 37°C. The digested DNAs were labeled by random priming with the Agilent Genomic DNA Labeling Kit Plus (Agilent technologies) for 2 hours at 37°C, according to the manufacturer’s instructions (Protocol v6.3, October 2010, Agilent technologies). Patients’ DNA and pooled normal control DNAs (reference) were labeled with Cy5-dUTP and Cy3-dUTP, respectively.
|
| |
|
| |
| Hybridization protocol |
Labeled products were purified with Microcon YM-30 filters (Millipore, Billerica, MA). Patient and normal control DNAs (reference) were mixed and hybridized with Human Cot I DNA (Invitrogen) at 65°C for 40 hours. The sandwiched slides were hybridized for 40 hours at 65°C with 15 rotations per minute and washed according to the Agilent protocol.
|
| Scan protocol |
Arrays were scanned with an Agilent DNA Microarray Scanner (G2565BA).
|
| Description |
Reference sample: Pool of six normal control DNAs from six healthy donors
|
| Data processing |
Log2 ratios were determined with Agilent Feature Extraction software (v 9.1.3.1). Results were visualized and analyzed with Agilent’s Genomic Workbench 5.0 software, and copy number aberrations were detected with the Aberration Detection Method 2 (ADM-2) algorithm using a threshold value of 6.0. This threshold value of the altered copy number was determined from the normal variation in the control hybridization.
|
| |
|
| Submission date |
Apr 18, 2011 |
| Last update date |
Oct 05, 2011 |
| Contact name |
Eric Pasmant |
| E-mail(s) |
eric.pasmant@gmail.com
|
| Organization name |
Paris Descartes University
|
| Department |
EA7331
|
| Street address |
Faculté de Pharmacie de Paris, 4 avenue de l’Observatoire
|
| City |
Paris |
| ZIP/Postal code |
75006 |
| Country |
France |
| |
|
| Platform ID |
GPL9777 |
| Series (1) |
| GSE28676 |
Molecular characterization of two unrelated patients ABCB4 deletions |
|
