Primary SCC carcinoma of the lung. Total RNA extracted from fresh-frozen tissue using Trizol and RNeasy clean-up.
Extracted molecule
total RNA
Label
Cy5
Description
Synthesis of the labelled first strand cDNA was conducted using Amersham CyScribe Post-Labelling kit with starting material of 20 ug of total RNA. The amino-allyl labeled dNTP mix was added to the reaction to generate amino-allyl labelled second strand cDNA. Following the hydrolysis reaction, single-stranded cDNA probes were purified using Amersham GFX columns. Dye coupling reactions were performed by mixing the cDNA samples with Amersham Cy3 (reference) or Cy5 (tumour) and incubating in the dark. The reactions were purified with Amersham GFX columns to remove the unincorporated/quenched dyes. After the purification samples were combined for hybridization, the labeled cDNAs were co-hybridized to 22K oligo microarrays. Slides were scanned on GMS418 confocal scanner (Agilent).
Data processing
Raw images were imported into Imagene V5.1 (BioDiscovery, CA, USA) to extract pixel intensities and to flag spots with poor/absent signal. The raw background subtracted signal median for each probe was imported into GeneSpring GX V7.3 (Agilent Technologies, Inc., CA, USA) for analysis. Data was normalized (Lowess: per spot and per chip; per chip to the 50th percentile) and probe signals filtered on pixel intensity (for both red and green channels any spots less than 20 units or greater than 65,000 units were excluded) and consistent spot morphology. Only spots flagged present in at least 80% of samples were included. For each probe, the logarithm to the base 2 of the ratio between the intensity in the tumor sample (red) channel and the reference (green) channel was used as the expression value for the probe.
