|
| Status |
Public on Dec 01, 2007 |
| Title |
Normal_colon_03_12April05 |
| Sample type |
RNA |
| |
|
| Source name |
colorectum, sigmoid
|
| Organism |
Homo sapiens |
| Characteristics |
Tissue: Colorectal mucosa
Location: (as per 'SOURCE')
|
| Biomaterial provider |
Flinders Medical Centre in collaboration with CSIRO P-Health Flagship
|
| Treatment protocol |
The colorectal specimens in this set were collected from a tertiary referral
hospital tissue bank in metropolitan Adelaide, Australia (Repatriation General Hospital and Flinders Medical Centre). The tissue bank and this project were approved by the Research and Ethics Committee of the Repatriation General Hospital and patient consent was received for each tissue studied. Following surgical resection, specimens were placed in a sterile receptacle and collected from theatre. The time from operative resection to collection from theatre was variable but not more than 30 minutes. Samples, approximately 125mm3 (5x5x5mm) in size, were taken from the macroscopically normal tissue as far from pathology as possible, defined both by colonic region as well as by distance either proximal or distal to the pathology. Tissues were placed in cryovials, then immediately immersed in liquid nitrogen and stored at -150degC until processing.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen samples were processed by the authors using standard protocols and commercially available kits. Briefly, frozen tissues were homogenized using a carbide bead mill (Mixer Mill MM 300, Qiagen, Melbourne, Australia) in the presence of chilled Promega SV RNA Lysis Buffer (Promega, Sydney, Australia) to neutralize RNase activity. Homogenized tissue lysates for each tissue were aliquoted to convenient volumes and stored -80degC. Total RNA was extracted from tissue lysates using the Promega SV Total RNA system according to manufacturers instructions and integrity
was assessed visually by gel electrophoresis.
|
| Label |
biotin
|
| Label protocol |
To measure relative expression of mRNA transcripts, tissue RNA samples were analyzed using Affymetrix HG U133 Plus 2.0 GeneChips (Affymetrix, Santa Clara, CA USA) according to the manufacturers protocols. Biotin labeled cRNA was prepared using 5ug (1.0 ug/uL) total RNA
(approx. 1 ug mRNA) with the One-Cycle cDNA kit (incorporating a T7-oligo(dT) primer) and the GeneChip IVT labeling kit. In vitro
transcribed cRNA was fragmented (20ug) and analyzed for quality control purposes by spectrophotometry and gel electrophoresis prior to hybridization.
|
| |
|
| Hybridization protocol |
An hybridization cocktail was prepared with 15ug of cRNA (0.5 ug/uL) and hybridized to HG U133 Plus 2.0 microarrays for 16h at 45degC in an Affymetrix Hybridization Chamber 640. Each cRNA sample was spiked with standard prokaryotic hybridization controls for
quality monitoring.
|
| Scan protocol |
Hybridized microarrays were stained with streptavidin phycoerytherin and washed with a solution containing biotinylated anti-streptavidin antibodies using the Affymetrix Fluidics Station 450. Finally, the stained and washed microarrays were scanned with the
Affymetrix Scanner 3000.
|
| Description |
Normal_colon_03_12April05.csv
|
| Data processing |
The Affymetrix software package was used to transform raw microarray image files to digitized format. As for the Discovery set above, gene expression levels for the this data set were calculated using MAS 5.0 (Affymetrix), scaled to 100 for quality control purposes and with the RMA normalization algorithm for expression data.
|
| |
|
| Submission date |
Oct 06, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Lawrence C LaPointe |
| E-mail(s) |
[email protected]
|
| Organization name |
Clinical Genomics
|
| Street address |
11 Julius Ave
|
| City |
North Ryde |
| State/province |
NSW |
| ZIP/Postal code |
2113 |
| Country |
Australia |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE9254 |
Normal human colorectal mucosa, cecum, ascending, transverse, sigmoid and rectum |
|
| Relations |
| Reanalyzed by |
GSE64985 |
| Reanalyzed by |
GSE119087 |
