tissue type: head and neck squamous cell carcinoma (HNSCC) anatomic site: OROPHARYNX gender: M hpv status: neg
Treatment protocol
All the patients were treated with concurrent chemoradiotherapy. Samples with microscopic tumor or tumor content <60% were excluded from further analysis.
Extracted molecule
total RNA
Extraction protocol
Guided by H&E stained slides, the region with the highest tumor content was cut from OCT blocks, pulverized using CryoPrep (Covaris, Woburn, MA) and homogenized in a lysis buffer from an AllPrepRNA/DNA/Protein Mini kit (Qiagen, Valencia, CA) or from a RNA/DNA/Protein Purification Kit (Norgen Biotek, Thorold, Canada) using an Ultrasonicator (Covaris, Woburn, MA). DNA, RNA and protein were isolated from each sample using the respective kit and following manufacturer’s protocol. RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). If DNA contamination was seen, RNAs were treated with TURBO DNA-free (Ambion, CA) to remove genomic DNA, and subsequently quantitated using a spectrophotometer. Only RNAs with integrity number (RIN) ≥7.5, a 260/280 ratio ≥1.8 and a 260/230 ratio ≥1.8 (≥1.1 for a few RNAs) were labeled for expression arrays.
Label
Cy3
Label protocol
Labeling for Agilent 4x44K v2 expression arrays was done using a Quick Amp Labeling Kit one-color according to the manufacturer’s protocol using 200ng of starting material.
Hybridization protocol
Expression arrays were performed at the Argonne National Laboratory in an ozone-controlled atmosphere.
Scan protocol
Expression arrays were performed at the Argonne National Laboratory in an ozone-controlled atmosphere.
Description
7029262
Data processing
The scanned images were analyzed with Feature Extraction Software 10.7.3.1. Raw data were background corrected, quantile normalized and log2 transformed.