|
| Status |
Public on May 28, 2010 |
| Title |
Primary Lung Adenocarcinoma_Sample_A67 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Universal Reference RNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: pool of 10 cell lines
sample type: reference
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The Universal Reference RNA was commercially available (Stratagene).
|
| Label |
Cy3
|
| Label protocol |
Synthesis of the labelled first strand cDNA was conducted using Amersham CyScribe Post-Labelling kit with starting material of 20 ug of total RNA. The amino-allyl labeled dNTP mix was added to the reaction to generate amino-allyl labelled second strand cDNA. Following the hydrolysis reaction, single-stranded cDNA probes were purified using Amersham GFX columns. Dye coupling reactions were performed by mixing the cDNA samples with Amersham Cy3 (reference) or Cy5 (tumour) and incubating in the dark. The reactions were purified with Amersham GFX columns to remove the unincorporated/quenched dyes.
|
| |
|
| Channel 2 |
| Source name |
Tumour sample A67, NARLC-AC
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: primary lung adenocarcinoma
lung cancer type: non-asbestos-related lung cancer-adenocarcinoma (NARLC-AC)
gender: male
age (yrs): 71
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from fresh-frozen tissue using Trizol (Invitrogen) and RNeasy (Qiagen) clean-up.
|
| Label |
Cy5
|
| Label protocol |
Synthesis of the labelled first strand cDNA was conducted using Amersham CyScribe Post-Labelling kit with starting material of 20 ug of total RNA. The amino-allyl labeled dNTP mix was added to the reaction to generate amino-allyl labelled second strand cDNA. Following the hydrolysis reaction, single-stranded cDNA probes were purified using Amersham GFX columns. Dye coupling reactions were performed by mixing the cDNA samples with Amersham Cy3 (reference) or Cy5 (tumour) and incubating in the dark. The reactions were purified with Amersham GFX columns to remove the unincorporated/quenched dyes.
|
| |
|
| |
| Hybridization protocol |
After the purification, the samples were combined for hybridization. The labeled cDNAs were co-hybridized to 22K oligo microarrays.
|
| Scan protocol |
Slides were scanned on a GMS418 confocal scanner (Agilent).
|
| Description |
Sample_A67
|
| Data processing |
Raw images were imported into Imagene V5.1 (BioDiscovery, CA, USA) to extract pixel intensities and to flag spots with poor/absent signal. The raw background-subtracted signal median for each probe was imported into GeneSpring GX V7.3 (Agilent Technologies, Inc., CA, USA) for analysis. Data was normalized (Lowess: per spot and per chip; per chip to the 50th percentile) and probe signals filtered on pixel intensity (for both red and green channels any spots less than 20 units or greater than 65,000 units were excluded) and consistent spot morphology. Only spots flagged present in at least 80% of samples were included. For each probe, the logarithm to the base 2 of the ratio between the intensity in the tumor sample (red) channel and the reference (green) channel was used as the expression value for the probe.
|
| |
|
| Submission date |
Mar 31, 2010 |
| Last update date |
Mar 31, 2010 |
| Contact name |
Jill Everland Larsen |
| E-mail(s) |
j.larsen@uq.edu.au
|
| Phone |
+61731394110
|
| Fax |
+61731394957
|
| Organization name |
The Prince Charles Hospital
|
| Department |
Thoracic Medicine
|
| Lab |
Thoracic Research Lab
|
| Street address |
Rode Road, Chermside
|
| City |
Brisbane |
| State/province |
Queensland |
| ZIP/Postal code |
4032 |
| Country |
Australia |
| |
|
| Platform ID |
GPL3877 |
| Series (1) |
| GSE20875 |
ADAM-28: A potential oncogene involved in asbestos-related lung adenocarcinomas |
|
