|Application:||Gene expression microarray analysis|
|Number of samples:||128|
|Release date:||Oct 31 2011|
|Last update date:||Jun 12 2013|
|Dataset link||Biology of human melanoma|
Tissue samples melanomas were obtained at Hospital A. C. Camargo. All patients signed an informed consent and the study was approved by our internal ethics committee. Tissue samples obtained by surgery were snap-frozen in liquid nitrogen and biopsy samples were collected in RNAlaterTM (Ambion, Austin, TX). Non-tumor areas were removed by handy dissection and only samples represented by at least 70% of tumors cells within their natural stroma were selected for RNA extraction. Total RNA was extracted using RNeasy Midi-Kit® and amplified by a T7-based protocol. An indirect cDNA-labeling was performed as described by Cox et al. (2004). We used dual-label system in which two RNA samples (test and reference) were separately reverse-transcribed, labeled, mixed, and hybridized together to each array. Each sample was hybridized in duplicate, with dye swap between sample and reference. As reference RNA we used a pool of equal amounts of total RNA from 5 human cutaneous melanoma cell lines (SK-MEL 05; 17; 28; 37 and 188, kindly provided by Dr. Alan Houghton, MSKCC, New York, NY, USA). We used glass arrays containing 4.800 cDNA sequences (Brentani et al. 2003) were prepared in our lab with the aid of the Flexys robot17 (Genomic Solutions, Cambridgeshire, United Kingdom). The list of immobilized genes can be obtained at GEO, accession number GPL1930. Pre-hybridization, hybridization and washing were performed as previously described (Gomes et al. 2003; 2005), and slides were scanned on a laser scanner (ScanArray Express, PerkinElmer Life Sciences, Boston, MA). Data were extracted with ScanArray Express software (PerkinElmer Life Sciences, Boston, MA) using the histogram method.
Waleska Kerllen Martins
BMC Med Genomics