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.
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').
