Unknown,Transcriptomics,Genomics,Proteomics

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HNSCC samples vs. normal tonsil samples: reference design

ABSTRACT: transcription profiling of human head and neck squamous cell carcinoma (HNSCC) samples vs. normal tonsil samples using a two-color reference design experimental setting. Used to identify differentially expressed genes in tumor/normal samples, and compare the result to that of the same samples using the self-self hybridization experimental setting. Keywords: tumor/normal comparison 8 HNSCC tumor samples and 8 normal tonsil samples. One sample per array. Two-color reference design with a common reference sample (a modified version of the Stratagene Human Universal Reference) on each array.

ORGANISM(S): Homo sapiens

SUBMITTER: D Hayes

PROVIDER: E-GEOD-13398 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Surgically resected gastric adenocarcinoma samples and their paired disease-free (R0) marginal nonmalignant tissue were obtained from 14 patients with gastric adenocarcinoma. RNA was isolated using the RNAeasy Kit (Qiagen Valencia, CA) from 15 mg of tissue. The quality of total RNA and its integrity was assessed using the Bioanalyzer 2100 (Agilent, Palo Alto, CA) and RIN value (RNA Integrity Number) was recorded for all the samples for intact 18S and 28S rRNA. Total RNA (800 ng) from each sample was reverse transcribed and linear amplified using the low RNA input linear amplification kit (Agilent Technologies). After synthesis of the first and second strand of cDNA, the product was used in an in vitro transcription reaction to generate cRNAs in the presence of cyanine 3-labeled UTP (Cy3) for normal and cyanine 5-labeled UTP (Cy5) for tumor. Fragmented Cy3-labeled cRNA of the control sample were mixed with equal amounts of Cy5-labeled cRNA from the gastric tumor sample, and the mixtures were hybridized onto 44K whole human genome DNA microarrays (Agilent Technologies) for 17 hrs at 65M-BM-0C with constant rotation (10 rpm). Subsequently, the arrays were washed according to manufacturerM-bM-^@M-^Ys instructions. The slides were scanned using an Agilent microarray scanner (G2565AA), and the images were processed using the Agilent feature extraction software AFE 9.5. GeneSpring GX v10.1 (Agilent Technologies) was used to analyze the expression profiles obtained after microarray hybridization. The feature extraction files from all the samples were uploaded to GeneSpring. Following intensity-dependent normalization (Lowess), the data was subjected to statistical analysis. A two tailed studentM-bM-^@M-^Ys t-test was used to determine significance of the differences observed between the normal and tumor samples. Benjamini Hochberg algorithm was used to compute false discovery rates. Genes were filtered based on P value threshold of 0.001 and false discovery rate of less than 1%. Transcriptional profiling of normal vs. tumor tissues from 14 patients with gastric cancer. Two-color experiment.

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HNSCC samples vs. normal tonsil samples: reference design

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