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.
RNA was extracted from microdissected tissue using the Absolutely RNA MicroPrep Kit (Stratagene, CA, USA) and quantitated using the RiboGreen Quantitation Reagent (Molecular Probes, OR, USA) both according to the manufacturers instructions. RNA quality was assessed by running RNA isolated from each tissue on the Agilent 2100 Bioanalyser (Agilent Technologies, Victoria, Australia). This showed a degree of RNA degradation in all samples but 18S and 28S ribosomal bands were clearly distinguished in 39/46 (84.8%) of cases. RNA was subject to two rounds of amplification using the RiboAmp RNA amplification kit (Acturus, CA, USA) according to the manufacturer’s instructions. For samples with sufficient RNA available, 100ng was used in the initial amplification reaction. Lesser initial quantities (50ng or 20ng) were used from smaller samples.
Label
Cy3
Label protocol
Due to the antisense orientation of the amplified RNA samples, a labelling protocol based on a method published by Schlingemann et al. was used to generate fluorescent labelled antisense cDNA for hybridisation to sense-oriented oligonucleotide microarrays. Briefly, 1 microgram of amplified RNA was reverse transcribed using random primers (pdN6, Amersham Biosciences) followed by RNase H digestion. cDNA labelling by Klenow fragment and amino-allyl incorporation was performed using the Bioprime Kit with a modified protocol (see Supplementary Methods for detailed description of labelling protocol). The appropriate NHS ester Cy dye (Cy3 or Cy5, Amersham Biosciences) was then coupled to the test and reference samples.
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.
RNA was extracted from microdissected tissue using the Absolutely RNA MicroPrep Kit (Stratagene, CA, USA) and quantitated using the RiboGreen Quantitation Reagent (Molecular Probes, OR, USA) both according to the manufacturers instructions. RNA quality was assessed by running RNA isolated from each tissue on the Agilent 2100 Bioanalyser (Agilent Technologies, Victoria, Australia). This showed a degree of RNA degradation in all samples but 18S and 28S ribosomal bands were clearly distinguished in 39/46 (84.8%) of cases. RNA was subject to two rounds of amplification using the RiboAmp RNA amplification kit (Acturus, CA, USA) according to the manufacturer’s instructions. For samples with sufficient RNA available, 100ng was used in the initial amplification reaction. Lesser initial quantities (50ng or 20ng) were used from smaller samples.
Label
Cy5
Label protocol
Due to the antisense orientation of the amplified RNA samples, a labelling protocol based on a method published by Schlingemann et al. was used to generate fluorescent labelled antisense cDNA for hybridisation to sense-oriented oligonucleotide microarrays. Briefly, 1 microgram of amplified RNA was reverse transcribed using random primers (pdN6, Amersham Biosciences) followed by RNase H digestion. cDNA labelling by Klenow fragment and amino-allyl incorporation was performed using the Bioprime Kit with a modified protocol (see Supplementary Methods for detailed description of labelling protocol). The appropriate NHS ester Cy dye (Cy3 or Cy5, Amersham Biosciences) was then coupled to the test and reference samples.
Hybridization protocol
See label protocol
Scan protocol
Agilent scanner
Description
Case 9.1, Normal, Grade 3, Gene Expression experiment