|
Status |
Public on Dec 31, 2010 |
Title |
lobular carcinoma in situ -1 |
Sample type |
genomic |
|
|
Channel 1 |
Source name |
FFPE tissue
|
Organism |
Homo sapiens |
Characteristics |
tissue: lobular carcinoma in situ patient: 1
|
Extracted molecule |
genomic DNA |
Extraction protocol |
5 mm sections were cut from the FFPE samples and prepared for microdissection. LCIS and invasive components were identified and microdissected using laser capture microdissection on the PixCell II. Captured cells were incubated in 50 mL of Proteinase K extraction buffer (50 mm Tris, 1 mM EDTA, 0.5% Tween-20) for 72 hours at 55°C. Proteinase K was inactivated by incubation at 95°C for 10 minutes prior to use for PCR. Degenerate oligonucleotide primed (DOP) PCR was performed according to the usual protocol. Briefly, 10-100ng of DNA was amplified by Thermo Sequenase DNA Polymerase in a low stringency pre-amplification step (5 cycles) followed by regular PCR amplification in less stringent conditions. Several DOP-PCR reactions were pooled from each sample (to reduce the effect of bias on each sample) and cleaned by phenol:chloroform extraction prior to quantification and labeling. The resulting DNA was quantified using a DynaQuant 200 Fluorometer).
|
Label |
Cy5
|
Label protocol |
2-3 mg of pooled DOP-PCR products from each sample as well as an equal amount of reference DNA was labelled by random priming in 3 separate reactions with either Cy3 or Cy5.
|
|
|
Channel 2 |
Source name |
FFPE lymph nodes, pooled
|
Organism |
Homo sapiens |
Characteristics |
tissue: pooled lymph nodes
|
Extracted molecule |
genomic DNA |
Extraction protocol |
5 mm sections were cut from the FFPE samples and prepared for microdissection. LCIS and invasive components were identified and microdissected using laser capture microdissection on the PixCell II. Captured cells were incubated in 50 mL of Proteinase K extraction buffer (50 mm Tris, 1 mM EDTA, 0.5% Tween-20) for 72 hours at 55°C. Proteinase K was inactivated by incubation at 95°C for 10 minutes prior to use for PCR. Degenerate oligonucleotide primed (DOP) PCR was performed according to the usual protocol. Briefly, 10-100ng of DNA was amplified by Thermo Sequenase DNA Polymerase in a low stringency pre-amplification step (5 cycles) followed by regular PCR amplification in less stringent conditions. Several DOP-PCR reactions were pooled from each sample (to reduce the effect of bias on each sample) and cleaned by phenol:chloroform extraction prior to quantification and labeling. The resulting DNA was quantified using a DynaQuant 200 Fluorometer).
|
Label |
Cy3
|
Label protocol |
2-3 mg of pooled DOP-PCR products from each sample as well as an equal amount of reference DNA was labelled by random priming in 3 separate reactions with either Cy3 or Cy5.
|
|
|
|
Hybridization protocol |
Labelled products were mixed in appropriate combinations in DIG Easy Hyb hybridization buffer and hybridized for 16-24 hours at 37°C to the human 19K arrays from the University Health Network Microarray Centre (Toronto, Canada) which contains 19008 characterized and unknown human genes and/or ESTs. Slides were rinsed in 0.1X SSC to remove the cover slips and then washed 3 times in 0.1X SSC/0.1% SDS for 10 minutes at room temperature. Slides were rinsed once in 1X SSC and twice in 0.1X SSC and centrifuged to dryness.
|
Scan protocol |
Arrays were scanned using the GenePix 4000A scanner (Axon Instruments, USA). The PMT gain for each laser was adjusted to give an average ratio of Cy3 to Cy5 of 1.0 and to minimize the number of saturated pixels. Images were then analyzed using the GenePix Pro 3.0 software.
|
Description |
lcis1a.gpr lcis1b.gpr
|
Data processing |
The resulting data were normalized using the printTipLoess algorithm in Bioconductor genomic analysis software package available on line at: http://www.bioconductor.org/. The data was normalized within each subarray as well as for the entire array. The values for the foreground and background for the duplicate spots were first averaged. Then the gene amplification level was calculated as the log base 2 of the ratio of the test to normal of the differences between foreground and background. The gene amplification level for each ‘fluor-flip’ experiment was averaged and filtered to exclude cDNA clones without mapping info.
|
|
|
Submission date |
Feb 22, 2010 |
Last update date |
Dec 31, 2010 |
Contact name |
Susan Done |
E-mail(s) |
sdone@uhnres.utoronto.ca
|
Organization name |
University Health Network
|
Street address |
200 Elizabeth Street, 11E444
|
City |
Toronto |
State/province |
ON |
ZIP/Postal code |
M5G2C4 |
Country |
Canada |
|
|
Platform ID |
GPL10093 |
Series (1) |
GSE20474 |
aCGH study of lobular carcinoma in situ (LCIS) and invasive lobular carcinoma (ILC) |
|