Strain; C57BL/6J, Type; WT, Gender; Male, Age; 10-12 wks, Tissue; Mucosal tissues from the rectum, The time of sacrifice; 16 days after the isolation stress. Equivalent amount of total RNA from rectum mucosa of 5 control mice were mixed equally and used for microarray.
Treatment protocol
Five brother mice were group-housed in one cage after weaning until 10-12 weeks age. Then, 3 of them were separated individually in an independent cage for the isolation stress. 16 days after the isolation stress, they were sacrificed under anesthetized with diethyl ether at 10:00 AM.
Extracted molecule
total RNA
Extraction protocol
The mucosal tissues from the rectum were incubated immediately after the collection with RNAlater stabilization reagent for 24 hours and stored at -80℃ until analysis. The rectum was considered as ranged in 7 mm total length from the anus. Total RNA was isolated following Qiagen's RNA isolation protocol (RNeasy Mini kit; Qiagen P/N 74104, Hilden, Germany). Contaminating DNA was removed using an RNase-free DNase set (Qiagen P/N 79254) during the process of RNA purification. The quality of the purified RNA applicable for microarray analysis was assessed by Agilent 2100 Bioanalyzer using an RNA 6000 Nano Labchip kit (Agilent Technologies).
Label
Cy3
Label protocol
Sample labeling, array processing and scanning, and data extraction were performed as described in One-Color Microarray-Based Gene Expression Analysis – Protocol. These protocols are available for download from Agilent's DNA Microarray Manuals website. The microarrays used were Agilent’s Whole Mouse Genome Oligo Microarrays (P/N G4122F). Probe sequence information is publicly available at Agilent's website. Labeled cRNAs were generated from 400 ng of total RNA using Agilent’s Low RNA Input Linear Amplification Kit (P/N 5188-5339).
Hybridization protocol
Hybridizations were performed using GE Hybridization Kit (P/N 5188-5242) and GE Wash Buffer 1 and 2 (P/N 5188-5325, -5326) as following Agilent's protocol.
Scan protocol
Microarrays were scanned using Agilent’s DNA Microarray Scanner BA (P/N G2565BA) and data extracted using Agilent's Feature Extraction software, version 9.5 (P/N G2567AA).
Description
The data from each "spot ID" of rectum mucosa of 5 isolated WT mice were subdivided by those of colonic mucosa of 5 control WT mice for all present spots.
Data processing
Data were processed using Agilent’s GeneSpring® GX software, version 7.3 (P/N G1745). Data were transformed by setting all measurements less than 0.01. Data points that did not have detectable signal and those that represent microarray controls were labeled as Absent, those representing either non-uniform or saturated features were labeled as Marginal, and all remaining data points were labeled as Present. All data points were 50 percentile scaled using the 50 percentile of signal intensity value for data points labeled as Present. Then the data from each "spot ID" of channel 1 was subdivided by channel 2.
Effects of psychological stress on gene expression in the mouse rectum.
Data table header descriptions
ID_REF
VALUE
Local background subtracted intensity data after percentile-scaling each array (Present only) between “Rectum, WT, stress” and “Colon, WT, control” was subdivided by that of “Colon, WT, control”. Only features that passed following filtering condition are shown: The raw signals and flags were more than 100 and present, respectively, at least in half (4 of 8) experiments.
Flag
Data points that did not have detectable signal and those that represent microarray controls were labeled as Absent, those representing either non-uniform or saturated features were labeled as Marginal, and all remaining data points were labeled as Present.
