Supplementary Material and Methods

Detection of ERa, ERb, PR, HER2 and Ki67, by reverse transcription (RT) and quantitative PCR (qPCR)

The same method was applied to human tumors and xenograft materials. First–strand cDNA synthesis was performed with 1 µg total RNA using Superscript II reverse transcriptase (RT, Invitrogen Corporation) in a final volume of 20 µL as previously described

Briefly, quantitative PCR analysis of ERa ERb, PR, HER2, and Ki67 transcripts as target genes and RPLPO (also known as 36B4) and TATA Box binding protein (TBP) as reference genes was performed on 6.25 ng samples cDNA in independent duplicate. The thermal cycling conditions comprised an initial denaturation step at 95°C for 10 min, then 45 cycles at 95°C for 15 sec, and 65°C for 1 min. All PCR reactions were done using the ABI Prism 7900 Sequence detection System (Applied Biosystems Inc., Forster City, USA). The nucleotide and probe sequences and the conditions of PCR have been previously described [1] [18]. Primer and fluorescent probes genes were designed from published sequences using primer Express software (Applied Biosystems Inc.). All genes were strictly blasted to obtain a specific human sequence and their specificity was initially assessed by the absence on normal mouse tissue. Human breast cancer cell lines T47D and MDA-MB 231 cDNA were used to generate 7 points standard curves for each gene [1] [18]. Target quantities were normalized to each of the reference genes and calibrated using the second point of each standard curve. Final results were expressed as N-fold differences in target gene expression relative to the reference genes and the calibrator and are expressed as :

E target (Ct calibrator – Ct sample) /E reference gene (Ct calibrator – Ct sample),

where E is the efficiency of PCR measured using the slope of the calibration curve, and Ct is the cycle threshold. No Reverse-transcription Controls (NTC) were included in each batch of samples. Only cases with exploitable data obtained for the 2 reference genes and the 5 target genes were submitted to further statistical analysis.Absolute quantification was done. Thresholds for a positive expression were defined by protein quantification for hormone receptors as previously described [2], by FISH for HER2 and by mitotic index for Ki67.

BAC array CGH

Our BAC array CGH analysis method has been previously reported [3]. Briefly, genomic DNA from patient tumors and xenografts at different passages was hybridized on glass slides bearing mapped sequenced BAC clones, at a resolution of 0.5 Mb all along the genome [3,4]. Each clone was spotted in quadruplicate on slide prepared by IntegragenTM (Evry, France). 1.5 µg of DNA sample were digested with DpnII enzyme (Ozyme), and labeled by random priming using a Bioprime DNA labeling kit (Invitrogen) with the appropriate cyanine dye (Cy3 or Cy5; Perkin-Elmer). The control and test DNAs were co-precipitated with Cot-1 DNA (Invitrogen), denatured, and resuspended in hybridization buffer. After 24 h hybridization, slides were washed with SDS and saline citrate, dried and scanned with a GenePix 4000B scanner (Axon Instruments Inc., Union City, CA, USA). Image analysis was done with Genepix 5.1 software (Axon) [5]. Any BAC with less than two replicates flagged for not fulfilling qualitative spot criteria was excluded. The MANOR algorithm was used to perform normalization [5]. Spots showing a low signal-to-noise ratio or poor replicate consistency were discarded. Status assignment (loss, normal, gain and amplification of chromosome copy number) was performed by using the GLAD algorithm [6]


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