DCIS foci were isolated from 10 micrometer serial frozen tissue sections by laser capture microdissection (PALM Microlaser Technologies AG, Bernried, Germany), guided by interval sections stained with haematoxylin and eosin. In addition, LCIS and co-existing areas of atypical ductal hyperplasia (ADH), proliferative disease without atypia (PDWA) and benign epithelium were sampled from a proportion of cases. For in situ lesions care was taken to capture pure intraduct cell populations. Benign epithelium samples were lobular tissue collected with intralobular stroma.
DNA was extracted from laser microdissected material using the QIAamp DNA Micro Kit (Qiagen Inc., Valencia, CA, USA) and quantitated using the PicoGreen dsDNA Quantitation Reagent (Molecular Probes) both according to the manufacturers instructions. Ten nanograms of DNA was amplified using the GenomiPhi DNA Amplification Kit (Amersham Biosciences, Piscataway, NJ, NY) according to the manufacturer’s instructions.
Label
Cy3
Label protocol
DNA was labelled with the Bioprime Kit (Invitrogen) using a modified protocol. Briefly, 3 micrograms of amplified test and reference DNA were directly labelled with either Cy3-dUTP or Cy5-dUTP (see Supplementary Methods for detailed description of labelling protocol). Samples were hybridised to the same oligonucleotide microarrays used for gene expression profiling.
DCIS foci were isolated from 10 micrometer serial frozen tissue sections by laser capture microdissection (PALM Microlaser Technologies AG, Bernried, Germany), guided by interval sections stained with haematoxylin and eosin. In addition, LCIS and co-existing areas of atypical ductal hyperplasia (ADH), proliferative disease without atypia (PDWA) and benign epithelium were sampled from a proportion of cases. For in situ lesions care was taken to capture pure intraduct cell populations. Benign epithelium samples were lobular tissue collected with intralobular stroma.
DNA was extracted from laser microdissected material using the QIAamp DNA Micro Kit (Qiagen Inc., Valencia, CA, USA) and quantitated using the PicoGreen dsDNA Quantitation Reagent (Molecular Probes) both according to the manufacturers instructions. Ten nanograms of DNA was amplified using the GenomiPhi DNA Amplification Kit (Amersham Biosciences, Piscataway, NJ, NY) according to the manufacturer’s instructions.
Label
Cy5
Label protocol
DNA was labelled with the Bioprime Kit (Invitrogen) using a modified protocol. Briefly, 3 micrograms of amplified test and reference DNA were directly labelled with either Cy3-dUTP or Cy5-dUTP (see Supplementary Methods for detailed description of labelling protocol). Samples were hybridised to the same oligonucleotide microarrays used for gene expression profiling.
