|
| Status |
Public on Aug 01, 2012 |
| Title |
21446-21N |
| Sample type |
RNA |
| |
|
| Source name |
Colon Tissue from patient with Crohn's disease
|
| Organism |
Homo sapiens |
| Characteristics |
disease: Chron's Disease
dategroup of array: A
cd68 expression: High
nos2 expression: High
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Tissues were flash frozen immediately after surgery.RNA from frozen tissue samples was extracted using standard TRIZOL (Invitrogen) protocol.
|
| Label |
Biotin
|
| Label protocol |
Processed by using a method of direct detection of the biotin-containing transcripts by streptavidin-Alexa647 conjugate.
|
| |
|
| Hybridization protocol |
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 |
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 |
CD68 and NOS2 were measured by qRT-PCR and dichotomized as high/low based on median expression levels Crohn's disease and Ulcerative Colitis separately. Missing values were dropped from analysis.
|
| Data processing |
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. 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. LOESS (Locally Weighted Scatterplot Smoothing) normalization was performed using the R software package and all replicate spots were averaged. Batch effects caused by day to day variability was removed using Partek Genomics Suite.
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| |
|
| Submission date |
Jun 02, 2011 |
| Last update date |
Aug 01, 2012 |
| Contact name |
Aaron Schetter |
| E-mail(s) |
schettera@mail.nih.gov
|
| Organization name |
National Cancer Institute
|
| Department |
Laboratory of Human Carcinogenesis
|
| Street address |
37 Convent Drive, Building 37, Room 3050
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL13681 |
| Series (1) |
| GSE29703 |
Association of microRNAs with CD68 and NOS2 in IBD tissues. |
|
