|
| Status |
Public on Feb 01, 2009 |
| Title |
IDC3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source Name |
IDC without known metastases into axillary lymph nodes
|
| Organism(s) |
Homo sapiens |
| Characteristics |
Microdissected sections of fresh fixed paraffin embedded tissue
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA extracted using a QIAGEN DNA Mini Kit (QUIAGEN Inc., Canada) after a 72 hr period in lysis buffer at 56°C. DNA from 12 normal lymph nodes was prepared similarly and pooled for the reference sample. The single cell comparative genomic hybridization protocol (SCOMP) was used for genome amplification
|
| Label |
Cy5
|
| Label protocol |
1.0μg of amplified test and reference DNA was mixed with 21.5μl ddH2O and 20μl of 2.5X Random Primer/Reaction Mix Buffer (Invitrogen, www.invitrogen.com), boiled for 5 min. and 5μl of ‘low C’ dNTP mix with either 2.5μl Cy5-dCTP (Amersham Biosciences, USA) for test or Cy3 for the reference DNA with 1μl of Klenow solution, then incubated at 37°C for 2 hrs. The reaction was stopped by adding EDTA, then 450μl of TE pH 7.4 added and the mix loaded onto a micron-30 filter (Fisher/Millipore), spun for 8-10 min. at 10,000 rpm, inverted and spun for 1 min to recover the purified probe.
|
| |
|
| Channel 2 |
| Source Name |
Pooled normal lymph nodes
|
| Organism(s) |
Homo sapiens |
| Characteristics |
Microdissected sections of fresh fixed paraffin embedded tissue
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA extracted using a QIAGEN DNA Mini Kit (QUIAGEN Inc., Canada) after a 72 hr period in lysis buffer at 56°C. DNA from 12 normal lymph nodes was prepared similarly and pooled for the reference sample. The single cell comparative genomic hybridization protocol (SCOMP) was used for genome amplification
|
| Label |
Cy3
|
| Label protocol |
1.0μg of amplified test and reference DNA was mixed with 21.5μl ddH2O and 20μl of 2.5X Random Primer/Reaction Mix Buffer (Invitrogen, www.invitrogen.com), boiled for 5 min. and 5μl of ‘low C’ dNTP mix with either 2.5μl Cy5-dCTP (Amersham Biosciences, USA) for test or Cy3 for the reference DNA with 1μl of Klenow solution, then incubated at 37°C for 2 hrs. The reaction was stopped by adding EDTA, then 450μl of TE pH 7.4 added and the mix loaded onto a micron-30 filter (Fisher/Millipore), spun for 8-10 min. at 10,000 rpm, inverted and spun for 1 min to recover the purified probe.
|
| |
|
| |
| Hybridization protocol |
Labeled products were combined, 80μg of Human Cot-1 DNA and 200mg of yeast tRNA (Invitrogen) were added. The mixture was concentrated in a speed-vac at 37°C down to 10μl and resuspended in 80μl of DIG Easy Hybe buffer (Roche, Switzerland), denatured at 100°C for 1.5 min., cooled on ice and incubated for 30 min at 37°C, then hybridized to 19K human cDNA arrays (University Health Network Microarray Centre, Toronto, Canada, www.microarrays.ca) for 16 hours at 42°C.
|
| Scan protocol |
GenePix 4000B slide scanner and GenePix Pro 6.0 software package (Molecular Devices, California, USA)
|
| Description |
run in duplicate
|
| Data processing |
Samples run in duplicate, Log2 ratios of duplicates averaged, filtered to exclude cDNA clones without mapping info and those spots flagged as anomalous, cenetered by the median and scale normalized.
|
| |
|
| Submission date |
Feb 21, 2008 |
| Contact name |
Susan Done |
| E-mail(s) |
sdone@uhnres.utoronto.ca
|
| Organization name |
Ontario Cancer Institute
|
| Street address |
610 University Ave
|
| City |
Toronto |
| ZIP/Postal code |
M5G2M9 |
| Country |
Canada |
| |
|
| Platform ID |
GPL6514 |
| Series (1) |
| GSE10600 |
aCGH study of invasive duct carcinoma (IDC) of the breast without axillary lymph node LN and with sentinel LN metastasis |
|
