|
| Status |
Public on Sep 21, 2009 |
| Title |
Normal Lung_NL25 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Universal Reference RNA
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Human Universal Reference RNA (Stratagene)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRizol following manufacturer's instructions
|
| 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 (Normal Lung) and incubating in the dark. The reactions were purified with Amersham GFX columns to remove the unincorporated/quenched dyes.
|
| |
|
| Channel 2 |
| Source name |
Normal Lung Sample 25
|
| Organism |
Homo sapiens |
| Characteristics |
gender: M
% predicted fev1: 59.2
% predicted kco: 67.9
age: 65
pack years: 40
age quit smoking: 61
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRizol following manufacturer's instructions
|
| 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 (Normal Lung) and incubating in the dark. The reactions were purified with Amersham GFX columns to remove the unincorporated/quenched dyes.
|
| |
|
| |
| Hybridization protocol |
Amersham hybridization protocol was used
|
| Scan protocol |
GMS418 confocal scanner was used according to manufacturer’s instructions
|
| Description |
After the purification samples were combined for hybridization, the labeled cDNAs were co-hybridized to 22K oligo microarrays.
|
| 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) 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 50% 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 |
Aug 21, 2009 |
| Last update date |
Sep 21, 2009 |
| Contact name |
Santiyagu Mary Savarimuthu Francis |
| E-mail(s) |
ss.francis@uqconnect.edu.au
|
| Phone |
07-31394110
|
| Fax |
07-31394957
|
| Organization name |
The University of Queensland
|
| Department |
School of Medicine
|
| Lab |
Thoracic Laboratory
|
| Street address |
Level 1, Clinical Sciences Building, The Prince Charles Hospital, Rode Road
|
| City |
Brisbane |
| State/province |
Queensland |
| ZIP/Postal code |
4032 |
| Country |
Australia |
| |
|
| Platform ID |
GPL3877 |
| Series (1) |
| GSE17770 |
Expression Profiling Identifies Genes Involved in Emphysema |
|
