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Sample GSM193887 Query DataSets for GSM193887
Status Public on Nov 24, 2008
Title Case 9.1, Normal, Grade 3, Gene Expression experiment
Sample type RNA
 
Channel 1
Source name Case 9.1, Normal, Grade 3, Gene Expression experiment
Organism Homo sapiens
Characteristics Sample Type:Normal;Grade:3
Extracted molecule total RNA
Extraction protocol 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.
 
Channel 2
Source name Stratagene Universal Human Reference RNA
Organism Homo sapiens
Characteristics Stratagene Universal Human Reference RNA
Extracted molecule total RNA
Extraction protocol 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
Data processing Loess-normalized log2 ratio
 
Submission date May 23, 2007
Last update date Nov 18, 2008
Contact name Sean Davis
E-mail(s) sdavis2@mail.nih.gov
Phone 301-435-2652
Organization name National Cancer Institute
Lab Genetics Branch
Street address 37 Convent Drive, Room 6138
City Bethesda
State/province MD
ZIP/Postal code 20892
Country USA
 
Platform ID GPL5326
Series (1)
GSE7882 Gene Expression and Comparative Genomic Hybridization of Ductal Carcinoma In Situ of the Breast

Data table header descriptions
ID_REF ID in platform
Red Intensity Background-subtracted Red Intensity
Green Intensity Background-subtracted Green Intensity
VALUE Log2 Ratio

Data table
ID_REF Red Intensity Green Intensity VALUE
1 298.2 132.7 2.6261288409111
2 29136.9 47274 0.0270156679614884
3 161.2 196.1 0.316347292922164
4 329.8 258.9 3.21574637388967
5 2249.7 1866.5 0.947508406031359
6 161.6 237.5 -2.03498488206824
7 1116.8 556.3 0.60116598849901
8 17857.7 32587.5 -0.256659585370690
9 4030.2 12785.8 0.646761492454762
10 547.2 2777.7 0.201938004299812
11 73.6 51.3 -0.228509768829539
12 2906.2 1957.1 -0.766884566996573
13 237.3 359.1 1.91112448326685
14 182.2 284.2 -0.0777378084990928
15 255.9 102.8 1.24911922333384
16 1233 653.4 1.69794893859383
17 577.3 5497.2 0.946297896574187
18 422 288.7 1.60628250090843
19 3995.9 18093.5 1.56902971258453
20 67.1 41.6 2.01814633515670

Total number of rows: 36288

Table truncated, full table size 1258 Kbytes.




Supplementary file Size Download File type/resource
GSM193887.txt.gz 581.3 Kb (ftp)(http) TXT
Processed data included within Sample table

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