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).
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
Hybridization were replicated with dye swap (see NCG22_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 the normalized Log (base 2) ratio of the median of Channel 1 (635 nm) to Channel 2 (532 nm)
