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Sample GSM1678973 Query DataSets for GSM1678973
Status Public on Dec 08, 2016
Title P13
Sample type RNA
 
Source name primary tumor
Organism Mus musculus
Characteristics source: tumor center tissue
Treatment protocol Transgenic Balb-NeuT mouse model of breast cancer (untreated)
Growth protocol not applicable
Extracted molecule polyA RNA
Extraction protocol Laser microdissection (PALM MicroBeam from Carl Zeiss MicroImaging GmbH) was performed to dissect metastatic lesions from lung, primary tumors, epithelial layers of mammary glands of BALB-NeuT mice at the time point of ADH, and BALB/c mice at different age. Small pieces summing up to 100,000 um2 for each sample were catapulted into a cap with 10 ul paramagnetic oligo-dT bead suspension and lysis buffer. The extraction of mRNA and microarray experiments were performed as described previously (Klein et al., 2002).
Label Cy5
Label protocol After cDNA synthesis the 3'-end was labeled with dGTP, so that a 3'-oligo-dG flanking region was generated. In the fourth step the whole transcriptome was amplified sequence-independent by PCR with a single primer. cDNA libraries were tested for CD30 and housekeeping gene expression by RT-PCR.
 
Hybridization protocol cDNA was hybridized to Operon-Microarray-Chips (Eurofins MWG Operon, Alabama, USA). Sample labeling and array hybridization were performed as described previously (Hartmann CH, Klein CA. Gene expression profiling of single cells on large-scale oligonucleotide arrays. Nucleic acids research. 2006;34(21):e143).
Scan protocol Slides were scanned on a GenePix 4000A Array Scanner. Images were gridded and spots were quantified using GenePix Pro 4.1 software. Features and areas of obvious defects were manually flagged and excluded.
Data processing Gene expression analysis was performed for 10 normal, 7 ADH, 19 primary tumor and 7 lung metastatic tissue samples. Libraries chosen for gene expression analysis demonstrated good expression of GAPDH, beta-actin and CD30 . cDNA was hybridized to Operon-Microarray-Chips (Eurofins MWG Operon, Alabama, USA). Sample labeling and array hybridization were performed as described previously (Hartmann and Klein 2006). Microarray data pre-processing was performed in R using the limma package (Smyth and Speed, 2003). Raw probe intensities were background corrected by applying the ‘normexp’ method (Smyth and Speed, 2003). Analyses were restricted to cy5 intensities, since cy3 intensities did not capture fold changes correctly in spike-in experiments of single-cell expression assays (not shown). Loess normalization was used in M versus A plots of individual cy5 intensities of a given transcript in a given array and the median cy5 intensity across all arrays of the same transcript. Log2 ratios were calculated from the normalized intensities and quantile normalization was applied across arrays. All further analysis was based on normalized log ratios. Transcripts present at significantly different levels between groups were identified using standardized linear models as implemented in the limma package (Wettenhall and Smyth 2004). Transcripts were considered differentially expressed when their corresponding adjusted p-value (Benjamini, et al 2001) was p<=0.05.
 
Submission date May 08, 2015
Last update date Dec 09, 2016
Contact name Christoph Andreas Klein
Organization name University of Regensburg
Department Experimental Medicine and Therapy Research
Street address Franz-Josef-Strauß-Allee 11
City Regensburg
State/province Bavaria
ZIP/Postal code 93053
Country Germany
 
Platform ID GPL13188
Series (2)
GSE68681 Cell density, Her2 and progesterone signaling regulate dissemination of breast cancer cells [Gene expression]
GSE68683 Cell density, Her2 and progesterone signaling regulate dissemination of breast cancer cells

Data table header descriptions
ID_REF
VALUE Although the raw data (gpr-files) are two channel, only Cy5 signal was analyzed, the value in the matrix table was calculated according to the defined data processing procedure (see 'data processing').

Data table
ID_REF VALUE
2 -0.55180057
3 -0.180021948
4 0.082048491
6 -0.401458912
7 -0.562769021
8 -0.046206223
9 -0.005530833
10 -0.558561888
11 -1.33459224
12 -0.124729
13 -1.101369246
14 0.167634546
15 -0.134709541
16 -0.257981623
17 -0.81901319
18 -0.386856149
19 -0.354184189
20 0.012917215
21 -0.262098003
22 -0.121894621

Total number of rows: 36008

Table truncated, full table size 636 Kbytes.




Supplementary file Size Download File type/resource
GSM1678973_060725_32.gpr.gz 2.7 Mb (ftp)(http) GPR
Processed data included within Sample table

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