Dataset features

Specifications


Application: Gene expression microarray analysis
Number of samples: 36
Release date: Dec 31 2011
Last update date: Mar 22 2012
Access: Public
Diseases: Melanoma, Neoplasms
Chemicals: Melphalan
Dataset link Gene expression profile of murine cutaneous melanoma after systemic treatment with tumor necrosis factor (TNF) associated or not with melphalan (MEL).

Experimental Protocol


We used amplified RNA (aRNA) from 18 tumor samples (Control – 5 samples; Mel – 4 samples; Tnfa – 4 samples; and Mel+Tnfa – 5 samples). For replica hybridization with dye swap, aRNA from the tumors and from a pool composed of equal amounts of RNA extracted from the K1735M2, M2R, B16F10, and B16F1 melanoma cells were labeled with Alexa555 or Alexa647. Labeled aRNA samples were hybridized against a glass platform containing 16,128 immobilized sense oligonucleotides (Fox Chase Cancer Center, USA) corresponding to murine genes. Slides were pre-hybridized at 42ºC for approximately 6 hours in a solution containing Denhardt’s 5X (Ficoll 400 0.05g/mL, poli-vynil-pyrrolidone 0.05g/mL, bovine serum albumin 0.05g/mL), SSC (sodium saline citrate) 5X, 0.2% SDS (Sigma), and 1% BSA. For hybridization, we used a mix of 3.5µg of each labeled sample aRNA and reference aRNA. The hybridization solution contained Denhardt’s 5X, formamide 25% (Sigma), SSC 5X, SDS 0.1%, salmon sperm DNA 0.1mg/mL (GE Healthcare), Poly-A 0.1mg/mL (GE Healthcare), and Cot-1 0.1mg/mL (GE Healthcare), to a final volume of 100µL. Hybridizations were carried out on a Gene Tac hybridization station (Genomic Solutions) at 42ºC for about 16h. The slides were removed from the hybridization cassettes directly to a recipient containing a washing solution (SSC 2X, SDS 0.1%) at 42ºC, put in a slide rack, transferred to another recipient containing fresh solution, and washed under constant agitation at 42ºC for 5 min. After that, they were washed twice for 5min in a second wash solution (SSC 0.1X, SDS 0.1%) at room temperature and rinsed five times for 1min at room temperature with a SSC 0.1X solution. The slides were dried upside down in a centrifuge at 1500rpm for 5min. The slides were scanned with a confocal laser scanner (ScanArrayTM Express, Perkin-Elmer Life Sciences, USA), and the spot intensities processed with the help of the ScanArray Express program (Packard BioScience), with a 10µm resolution and a PMT of 60% for Alexa 555 and of 70% for Alexa 647. Each slide generated a data set for each channel corresponding to the dyes Alexa 555 and Alexa 647. The slides were scanned with a confocal laser scanner (ScanArrayTM Express, Perkin-Elmer Life Sciences, USA) with a 10µm resolution and a PMT of 60% for Alexa 555 and of 70% for Alexa 647, and data were extracted with ScanArray Express software (Packard BioScience) using the histogram method. Each slide generated a data set for each channel corresponding to the dyes Alexa 555 and Alexa 647. The Locally Weighted Scatter-plot Smoothing method (LOWESS), adjusted for linear and non-linear systematic variations, both of the intensity dependent type, mainly for low intensity spots, was employed for data normalization.

Repositories


GEO

GSE21559

ArrayExpress

E-GEOD-21559

BioProject

PRJNA126233

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Contact


Renato David Puga