Tissue samples and RNA isolation
Tissue samples: Intraperitoneal injection of the human breast cancer cell line mcf7 into severe combined immunodeficiency (SCID) mice resulted in the growth of a solid tumour mass [16–18] varying in diameter from 1 to 7 mm. Six to nine weeks post injection, mice were sacrificed by application of CO2, and the tumour mass was immediately frozen in liquid nitrogen. Human placental tissue was frozen as soon as possible. Total cellular RNA was isolated with the Trizol-method (TriFast, peqlab, Erlangen, Germany), following the manufacturers instructions, after homogenisation with a Mikro-Dismembrator S (Braun Biotech, Melsungen, Germany). The quality of the RNA samples was checked by the Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany). Only high-quality RNA samples (ratio 28S/18S rRNA > 1.8) were selected for the experiments.
The cDNA synthesis was performed using a cDNA Synthesis System Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturers instructions. The ds cDNA was purified by phenol/chloroform extraction  precipitated in 5 M NH4OAc  and resuspended in 8 μl RNase-free water.
In vitro transcription  was performed using the AmpliScribeTM T7 High Yield Transcription Kit (Epicentre Technologies, Madison, WI, USA) according to the manufacturers instructions with the exception of increasing the incubation time to 4 hours. The newly synthesized aRNA was extracted in phenol/chloroform/isoamylalcohol as described above. Unincorporated dNTPs were removed by chromatography with MobiSpin S-300 columns (MoBiTec GmbH, Göttingen, Germany). Ethanol precipitation was performed as described in  and the resulting pellet was resuspended in 20–50 μl RNase-free water.
Glass slides used for this study carried 7,347 breast specific cDNA clones selected from the Human UniGene 1 clone set (German Resource Centre for Genome Research, Berlin, Germany). The clones were amplified by PCR and spotted in a "replicate array" design on silane-prep™ slides (Sigma Diagnostics, St. Louis, USA) to produce 14,694 individual spots. The slides were rehydrated, and the DNA was denatured with boiling water treatment prior to washing with 0.2% SDS, MilliQ- H2O, 95% and 100% Ethanol. After the washing procedure, the microarrays were dried with compressed air.
RNA labelling and hybridisation
2 μg amplified antisense RNA (aRNA) were mixed with 500 ng random hexamer primers, incubated at 70°C for 10 min and cooled on ice. Alternatively, 10 μg total RNA were mixed with 500 ng (dT)17 primer, incubated at 70°C for 10 min and cooled on ice. The labelling reaction was performed in a 12.5 μl reaction volume using 2.5 μl 5x RT buffer (Invitrogen, Karlsruhe, Germany), 1.25 μl 0.1 M DTT (Invitrogen), 1 μl dNTP-mix (dGAT, 5 mM each), 0.5 μl dCTP (3 mM), 0.5 μl RNasin (40 U/μl), 0.5 μl Cy-3 or Cy-5 labelled dCTP (1 mM, Amersham Biosciences, Freiburg, Germany) and 1 μl Superscript II reverse transcriptase (100 U/μl, Invitrogen). The mixture was incubated for 1 h at 42°C, and the reaction was stopped by addition of 1.25 μl 50 mM EDTA (pH 8). The input RNA was removed by hydrolysis with 5 μl 1 M NaOH at 65°C for 10 min, followed by neutralisation with 1 μl 5 M acetic acid. Cy-3 and Cy-5 labelled probes were combined, precipitated with isopropanol, washed with 70% ethanol, air dried and resuspended in 30 μl hybridisation buffer. The hybridisation buffer consisted of 350 μl 2x DIG-EASY (Roche Diagnostics), 210 μl MilliQ-H2O, 70 μl 50x Denhardt's solution  and 70 μl Cot1-DNA (10 ng/μl). The 30 μl probe was heat denatured at 65°C for 2 min and hybridised to cDNA glass microarrays in a hybridisation chamber (Corning Inc., Acton, USA) over night at 37°C. After hybridisation the microarrays were washed at 25°C in a water bath with 1x SSC containing 0.1% SDS once for 15 min, and twice for 10 min. This was followed by washing twice with 0.1x SSC containing 0.1% SDS for 10 min. Washing steps were completed in 70% and 95% ethanol before the microarrays were dried.
The hybridised arrays were scanned with the GenePix 4000 B microarray scanner (Axon Instruments Inc., Union City, CA, USA), and the scanned images were analysed using GenePix Pro 4.0 software (Axon Instruments). Spot intensities were obtained by subtracting the median brightness of a region around the spot from the median of the region within.
For each of the datasets α ... η (table 2), the spot intensities were calibrated and transformed by the VSN method . Differences between the resulting values are referred to as "generalised log ratios". All 14.694 spots from each slide were analysed, without filtering or thresholding procedures.
The reproducibility of different amplification reactions was measured using the F statistic to compare the variance for each gene within and between groups of hybridisations using amplified samples. The F value is defined as the ratio of the between and within groups mean squares mSS
, whereas the mean square is defined as the sum of squares divided by the degrees of freedom:
denotes the measured value of the l th repetition of the k th amplification reaction for RNA transcript i. K denotes the number of all amplification reactions and L
the number of replicas in group k.