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Sample GSM211573 Query DataSets for GSM211573
Status Public on Jul 01, 2008
Title Large Congenital Melanocytic Naevi (CMN) : NCG21_1
Sample type RNA
 
Channel 1
Source name Large Congenital Melanocytic Naevi (CMN) : NCG21_1
Organism Homo sapiens
Characteristics Cells from Large Congenital Melanocytic Naevi. BRAF V600E mutated sample.
Biomaterial provider Dessars B; De Raeve L
Treatment protocol Cells from Large Congenital Melanocytic Naevi samples were isolated using a collagenase/dispase preparation (Morandini R., personal communication).
Growth protocol Cell cultures were performed with Ham-F10 basal medium supplemented with 9% FCS (Invitrogen, Carlsbad, CA, USA), L-Glutamine 1mM (Sigma-Aldrich, Saint-Louis, Missouri, USA), Penicilline G 100U/ml (Sigma-Aldrich, Saint-Louis, Missouri, USA), Kanamycine Sulfate 100µg/ml (Invitrogen, Carlsbad, CA, USA), Streptomycine Sulfate 100µg/ml (Invitrogen, Carlsbad, CA, USA), Choleratoxin 1nM (Sigma-Aldrich, Saint-Louis, Missouri, USA), BPE 40µg/ml, TPA 30nM (Sigma-Aldrich, Saint-Louis, Missouri, USA), IBMX 50µM (Sigma-Aldrich, Saint-Louis, Missouri, USA), Phosphoéthanolamine 100µM (Sigma-Aldrich, Saint-Louis, Missouri, USA), Hydrocortisone 0.5µM (Sigma-Aldrich, Saint-Louis, Missouri, USA), Ethanolamine 100µM (Sigma-Aldrich, Saint-Louis, Missouri, USA), all of these from the same batch. In order to prevent fibroblastic contamination, Geneticine (200 µg/ml, Carlsbad, CA, USA) was added at passage 1 and during 24 hours.
Extracted molecule total RNA
Extraction protocol RNA from melanocytic cells was extracted by ultracentrifugation in a guanidine isothiocyanate-cesium chloride gradient, followed by phenol-chloroform RNA extraction.
Label Cy5
Label protocol Double-stranded cDNA was synthesized from 1 µg of total RNA, followed by production of antisense RNA containing the modified nucleotide 5-(3-aminoallyl)-UTP using the Amino Allyl MessageAmpTM II aRNA Amplification kit (Ambion, Texas, USA). Label source :GE Healthcare Bio-Sciences, New Jersey, USA).
 
Channel 2
Source name Reference RNA : pool of 13 different foreskins
Organism Homo sapiens
Characteristics Pool of normal melanocytes cells from 13 different foreskins
Biomaterial provider Dessars B
Treatment protocol Cells from normal melanocytes obtained from 13 different foreskins were isolated using a collagenase/dispase preparation (Morandini R., personal communication).
Growth protocol Cell cultures were performed with Ham-F10 basal medium supplemented with 9% FCS (Invitrogen, Carlsbad, CA, USA), L-Glutamine 1mM (Sigma-Aldrich, Saint-Louis, Missouri, USA), Penicilline G 100U/ml (Sigma-Aldrich, Saint-Louis, Missouri, USA), Kanamycine Sulfate 100µg/ml (Invitrogen, Carlsbad, CA, USA), Streptomycine Sulfate 100µg/ml (Invitrogen, Carlsbad, CA, USA), Choleratoxin 1nM (Sigma-Aldrich, Saint-Louis, Missouri, USA), BPE 40µg/ml, TPA 30nM (Sigma-Aldrich, Saint-Louis, Missouri, USA), IBMX 50µM (Sigma-Aldrich, Saint-Louis, Missouri, USA), Phosphoéthanolamine 100µM (Sigma-Aldrich, Saint-Louis, Missouri, USA), Hydrocortisone 0.5µM (Sigma-Aldrich, Saint-Louis, Missouri, USA), Ethanolamine 100µM (Sigma-Aldrich, Saint-Louis, Missouri, USA), all of these from the same batch. In order to prevent fibroblastic contamination, Geneticine (200 µg/ml, Carlsbad, CA, USA) was added at passage 1 and during 24 hours.
Extracted molecule total RNA
Extraction protocol RNA from melanocytic cells was extracted by ultracentrifugation in a guanidine isothiocyanate-cesium chloride gradient, followed by phenol-chloroform RNA extraction.
Label Cy3
Label protocol Double-stranded cDNA was synthesized from 1 µg of total RNA, followed by production of antisense RNA containing the modified nucleotide 5-(3-aminoallyl)-UTP using the Amino Allyl MessageAmpTM II aRNA Amplification kit (Ambion, Texas, USA). Label source :GE Healthcare Bio-Sciences, New Jersey, USA).
 
 
Hybridization protocol Slides were incubated at 42°C for 60 min in the following preheated (42°C) solution: 5xSSC, SDS 0.1%, HEPES 24mM, BSA 0.1mg/ml. Slides were rinsed for 30s in deionized water followed by a centrifugation at 800 rpm for 10 min. Cyanine-labeled cRNA samples were mixed with 30 µg of human cot-1, incubated for 2 min at 100°C, centrifuged for 10 min at 13,000 rpm, and then kept at 65°C. After hybridization of the samples on the microarray slides overnight at 65°C, slides were washed twice for 2 min at 60°C with 2xSSC, 0.03% SDS, once 2 min at 60°C with 2xSSC, and finally briefly washed in 0.2xSSC at room temperature. They were dried by centrifugation at 800 rpm for 13 min and stored in the dark at room temperature.
Scan protocol Slides were scanned using a Molecular Devices 4000B laser scanner and expression levels were quantified using GenePix Pro 6.1 image analysis software (Axon Instruments, CA, USA). Image acquisitions were performed with automatic photomultiplier gains (PMT) adjustement. Artifact-associated spots were eliminated by both visual and software guided flags.
Description Hybrization were replicated with dye swap (NCG21_2)
Data processing A non-linear Lowess (locally weighted scatter plot) normalization method applied to each individual block (print-tip option) was carried out.
VALUE is Log (base 2) of the ratio of the median of Channel 1 (635 nm) to Channel 2 (532 nm)
 
Submission date Jul 19, 2007
Last update date Aug 14, 2011
Contact name Frederick Libert
E-mail(s) flibert@ulb.ac.be
Organization name ULB
Department IRIBHM
Lab C2-140
Street address 808 route de Lennik
City Brussels
ZIP/Postal code B-1070
Country Belgium
 
Platform ID GPL5621
Series (1)
GSE8525 Genotypic and gene expression studies in Congenital Melanocytic Nevi: insight into initial step of tumoral melanogenesis

Data table header descriptions
ID_REF
VALUE print-tip Loess normalized log2 ratio data (test/reference)
CH1_SIG_MEDIAN Median feature pixel intensity at 635 nm.; Scale: linear_scale
CH1_BKD_MEDIAN Local median background pixel intensity at 635 nm.; Scale: linear_scale
CH2_SIG_MEDIAN Median feature pixel intensity at 532 nm.; Scale: linear_scale
CH2_BKD_MEDIAN Local median background pixel intensity at 532 nm.; Scale: linear_scale

Data table
ID_REF VALUE CH1_SIG_MEDIAN CH1_BKD_MEDIAN CH2_SIG_MEDIAN CH2_BKD_MEDIAN
hCA045328 0.015 101 101 103 101
hCA045389 -0.107 106 102 169 202
hCA045395 0.178 135 118 163 168
hCA045423 -0.036 101 94 105 103
hCA045439 -0.041 112 98 188 239
hCA045498 -0.03 114 104 158 176
hCA045528 0.209 202 198 161 178
hCA045530 0.012 97 98 97 99
hCA045532 -0.066 97 91 107 103
hCA045541 -0.096 110 111 112 109
hCA045542 -0.003 96 98 97 98
hCA045589 0.012 115 115 114 117
hCA045599 0.106 137 121 173 181
hCA045609 -0.068 109 107 110 110
hCA045614 -0.007 106 106 105 105
hCA045640 -0.036 99 93 107 93
hCA045725 -0.138 101 93 134 143
hCA045740 -0.225 100 98 222 285
hCA045759 -0.102 91 92 104 111
hCA045765 -0.2 101 89 151 162

Total number of rows: 48958

Table truncated, full table size 1541 Kbytes.




Supplementary file Size Download File type/resource
GSM211573.gpr.gz 4.1 Mb (ftp)(http) GPR
Processed data included within Sample table

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