|
| Status |
Public on Feb 12, 2008 |
| Title |
Paired nontumorous tissue (12031n) |
| Sample type |
RNA |
| |
|
| Source name |
12031n
|
| Organism |
Homo sapiens |
| Characteristics |
Tissue: Colon
Disease state: paired nontumorous tissue
Individual: 12031
Clinical info: Follow up status: Died from Cancer
|
| Extracted molecule |
total RNA |
| Extraction protocol |
OSU - Tri-Reagent Protocol
Other: RNA isolation was performed using TRIZOL Reagent (Invitrogen, Carlsbad) according to manufacturer's instructions. Briefly, colon tissue was homogenized in TRIZOL reagent and chloroform was added and liquid phases were separated with centrifugation. The supernatant was collected and RNA was precipitated with isopropanol washed with 75% ethanol. RNA was resuspended in DEPC treated water and 5 micrograms were used for each microarray experiment.
|
| Label |
alexa 647
|
| Label protocol |
OSU - Labeling Protocol
Other: Processed by using a method of direct detection of the biotin-containing transcripts by streptavidin-Alexa647 conjugate.
|
| |
|
| Hybridization protocol |
OSU - Sample Hybridization Protocol
Qty. of labeled target: 5 ug of total RNA
Other: Labeled targets from 5ug of total RNA were used for hybridization on each miRNA microarray chip containing human and mouse miRNA genes. All probes on these microarrays are 40-mer oligonucleotides spotted by contacting technologies and covalently attached to a polymeric matrix. The microarrays were hybridized in 6x SSPE (0.9 M NaCl / 60 mM NaH2PO4 H20 / 8 mM EDTA, pH 7.4) / 30% formamide at 25C for 18 hr, washed in 0.75x TNT (Tris HCL/NaCl/Tween 20) at 37C for 40 min.
|
| Scan protocol |
OSU - Scanning Protocol
Other: Processed slides were scanned by using a PerkinElmer ScanArray XL5K Scanner, with the laser set to 635 nm, at Power 80 and PMT 70 setting, and a scan resolution of 10 um.
|
| Description |
No additional information.
|
| Data processing |
Data Processing Protocol
Calculation Method: The microarrays used for this analysis were pin-spotted microRNA microarrays (from the Ohio State University Comprehensive Cancer Center, version 2.0). Intensities of each spot were the median intensities of foreground. Each of 170 microarrays used for this study contained 11520 spots. All spots where foreground intensity was less than background were reassigned as NA (NA marks missing data spots). All spots flagged as deficient by the scanner were also reassigned as NA. All blank (no oligo) spots with high foreground intensity were reassigned as NA. Each microRNA oligo is represented by quadruplicate spots on these arrays as two distant pairs of two adjacent spots. If there were 0 or 1 NA for an oligo quadruple, and the means of the distant oligo pairs differed by > 1 on the log2 scale, all of the quadruplicate spots were reassigned as NA. If there were 2 NAs for an oligo quadruple and the two non-NA spot intensities differed by > 1 on the log2 scale, all of the quadruplicate spots were reassigned NA. If there were 3 NA spots for a quadruple, the final spot was reassigned as NA. In total, 1,082,689 of 1,958,400 spots were reassigned as NA using these methods. LOESS (Locally Weighted Scatterplot Smoothing) normalization was performed using the R software package. All data was then imported into BRB array tools version 3.5.0 for analysis and all replicate spots were averaged. There were originally 85 pairs (tumor and paired nontumorous tissue) of arrays used. One case that was originally identified as an incident colon carcinoma patient was later found to have been diagnosed as carcinoma in situ and was removed from the analysis leaving the study population of 84 subjects.
|
| |
|
| Submission date |
May 17, 2007 |
| Last update date |
Feb 12, 2008 |
| Contact name |
Curtis C Harris |
| E-mail(s) |
Curtis_Harris@nih.gov
|
| Phone |
(301) 496-2048
|
| Fax |
(301) 496-0497
|
| Organization name |
National Cancer Institute
|
| Department |
Center for Cancer Research
|
| Lab |
Laboratory of Human Carcinogenesis
|
| Street address |
Building 37, Room 3068A
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL4700 |
| Series (1) |
| GSE7828 |
MicroRNA profiles of 84 colon adenocarcinomas and paired nontumorous |
|
