uid: cca0746b2101488088257e2a88a9993c filenumber: 10 disease state: late stage OSCC tissue: tumor tumor stage: III
Treatment protocol
not applicable
Growth protocol
Tumor samples and the surrounding normal margins were collected from the oral squamous cell carcinoma (OSCC) patients. The tissues collected were immediately placed in RNA later solution (Qiagen) and further stored at -80⁰C till further use.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from the tissues using Qiagen Rneasy mini kit according to manufacturers protocol.
Label
biotin
Label protocol
labelling was performed according to the Affymetrix (Santa Clara, CA) GeneChip Whole Transcript (WT) Sense Target Labelling Assay manual
Hybridization protocol
microarrays were hybridized overnight with 5 mg biotin labelled ss-cDNA, and washed in fluidics station wash protocol: MES_EukGE-WS2v5_450
Scan protocol
Genechip scanner 3000 7G
Data processing
The input files were normalized with full quantile normalization (Irizarry et al 2003). For each input array, for each probe expression value, the array ith percentile probe value was replaced with the average of all array ith percentile points. Next, the 818,005 probes were manipulated into the analysis values as follows. Probes with GC count less than 6 and greater than 17 were excluded from the analysis. Probe scores were then transformed by taking the Base-2 Logarithm of 0 plus the probe score. Background CorrectionExon arrays do not use individual mis-match probes. Background is established from a pool of probes designed for that purpose. Background probes are stratified by CG content and are defined in the HumanGene10ST_antigenomic.bgp file. BGP files can also be downloaded from www.affymetrix.com. Each probe score was corrected for background by subtracting the median expression score of background probes with similar GC content. Probe-set Presence/Absence and the Removal of Non-expressed Probe-setsNon-expressed probes can cause tests for alternative splicing to find false positives (because they cause 'non-parallel' expression patterns across the gene). A probe-set is judged to be expressed above background if for any group: Integral from T0 to Infinity of the standard normal distribution < Significance (0.05) Where: T0 = Sqr(GroupSize) (T - P) / Sqr(Pvar), GroupSize = Number of CEL files in the group, T = Average of probe scores in probe-set, P = Average of Background probes averages of GC content, and Pvar = Sum of Background probe variances / (Number of probes in probe-set)^2,Hence we test that the average of probe-sets in a group is greater than the average expression of background probes of similar gc content as the probe-set probes as the center of background for the probe-set and derive its dispersion from the background probe-set variance.
