|
| Status |
Public on Jan 01, 2013 |
| Title |
RNA-UPPP-02 |
| Sample type |
RNA |
| |
|
| Source name |
normal
|
| Organism |
Homo sapiens |
| Characteristics |
sample identifier: Ctrl 102
sample type: normal
|
| Treatment protocol |
human surgical specimens, flash frozen
|
| Growth protocol |
human HNSCC and UPPP surgical samples
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
biotin
|
| Label protocol |
RNA from control and experimental samples were labeled using the Whole Transcript Sense Target Labeling protocol described by Affymetrix and reagent from Ambion and Affymetrix. Briefly, 100ng of total RNA was used to synthesize first strand cDNA using random oligonucleotides with T7 promoter as primer and the SuperScript Choice System (Invitrogen, Carlsbad, California). Following the double stranded cDNA synthesis, the double strand cDNA was purified using magnetic beads, and cRNA was generated through in vitro transcription. 10 ug of cRNA was then used to generate sense strand cDNA using random primer, dNTP-dUTP mix, and Superscript II reverse transcriptase (Invitrogen, Carlsbad, California). The resulting sense strand cDNA was then purified, fragmented using UDG and APE at 37C for 60 minutes, and terminal labeled with biotinylated nucleotide and terminal DNA transferase at 37C for 60 minutes.
|
| |
|
| Hybridization protocol |
The labeled sense strand DNA was hybridized to Affymetrix GeneChip miRNA 1.0 for 17hr at 45 C with constant rotation (60 rpm). Affymetrix Fluidics Station 450 was used to wash and stain the Chips, removing the non-hybridized target and incubating with a streptavidin-phycoerythrin conjugate to stain the biotinilated cDNA. The staining was further amplified using goat IgG as blocking reagent and biotinilated anti-streptavidin antibody (goat), followed by a second staining step with a streptavidin-phycoerythrin conjugate.
|
| Scan protocol |
Fluorescence was detected using the Affymetrix G3000 GeneArray Scanner and image analysis of each GeneChip was perfomed through the Affymetrix GeneChip Command Console version 3.4 (AGCC v3.4) software from Affymetrix.
|
| Data processing |
Analysis was performed in R with the oligo package, RMA was used for normalization; only H.sapiens probesets were retained for analysis.
|
| |
|
| Submission date |
Dec 16, 2011 |
| Last update date |
Jan 01, 2013 |
| Contact name |
Michael F Ochs |
| Organization name |
Johns Hopkins University
|
| Department |
Oncology Biostatistics
|
| Street address |
550 North Broadway, Ste 1103
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL8786 |
| Series (2) |
| GSE33232 |
Cancer Outlier Gene Profile Sets Elucidate Pathways and Patient-Specific Targets in Head and Neck Squamous Cell Carcinoma |
| GSE34496 |
miR-182 is Overexpressed in Head and Neck Squamous Cell Carcinomas Leading to Downregulation of Specific Transcripts |
|
