|Public on Mar 05, 2014
|Analysis 1: HPV Active - Biological replicate 8
|Oropharyngeal squamous cell carcinoma - HPV Active
race: European American
|Total RNA was isolated from fresh frozen tumor samples using a miniaturized mortar and pestle system (CryoGrinder™, CryoCooler™, OPS diagnostics LLC) and TRIzol® Reagent (Ambion®, by Life Technologies).
|Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using the Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with a NanoDrop 2000c Spectrophotometer.
|600 ng of Cy3-labelled cRNA (specific activity >8.6 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent-039494 SurePrint G3 Human GE v2 8x60K Microarray (G4858A-039494) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then immersed in acetonitrile and dried immediately by slowly removing the slide.
|Slides were scanned immediately after washing on an Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3 um, Dye channel is set to Green at 20 bit).
|re-analysis of US10130359_253949411321_S01_GE1_107_Sep09_2_4 sample in analysis 3, 5
|The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid 039494_D_F_20120628) to obtain background subtracted and spatially detrended Processed Signal intensities. Data was uploaded into Genespring version 11.5.1 where it was log2 transformed, quantile normalized and base line transformed using the median of all samples. Data was filted by flags in a way that at least 75% of the samples in any of the experimental groups had DETECTED flags. Then, differentially expressed genes were determined.
|Mar 04, 2014
|Last update date
|Mar 05, 2014
|University of South Carolina
|Department of Drug Discovery and Biomedical Sciences
|Functional Genomics Core
|715 Sumter Street, Room 617
|Gene expression profiling of HPV-active, HPV-inactive and HPV-negative oropharyngeal squamous cell carcinomas
|Gene expression profiles of oral and oropharyngeal cancers from European American and African American patients