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Sample GSM513685 Query DataSets for GSM513685
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)

Data table header descriptions
ID_REF
VALUE normalized log2 ratio (Cy5/Cy3)

Data table
ID_REF VALUE
1 -0.001725863
2 -1.14747148
3 -0.427641752
4 -0.947009079
5 -0.720116609
6 0.635767132
7 -0.621564327
8 0.284120034
9 -0.518836974
10 -0.411472495
11 -0.421910238
12 -0.520859197
13 -1.007281387
14 -0.798810733
15 -0.796418263
16 -0.796418263
17 -0.796418263
18 -0.29384996
19 -0.564875288
20 -0.152733904

Total number of rows: 10490

Table truncated, full table size 177 Kbytes.




Supplementary file Size Download File type/resource
GSM513685_lcis1a.gpr.gz 1.2 Mb (ftp)(http) GPR
GSM513685_lcis1b.gpr.gz 1.2 Mb (ftp)(http) GPR
Processed data included within Sample table

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