|Public on Apr 17, 2014
|WT, biological rep1
|DSS-treated colons derived from WT mice
genotype/variation: wild type
age: fourteen weeks
|Wild type (WT) and STAP-2-/- mice were administered 1.75% DSS for 3 days and sacrificed to isolate mRNA from colon tissue.
|Total RNA of the entire colon tissues, which was immediately stored in RNAlater solution at -80 °C (Life Technologies, Carlsbad, CA), was purified using TissueLyser at 20 Hz and RNeasy kit (QIAGEN, Venlo, Netherlands) according to the manufacturer's instructions.
|One hundred nanogram of total RNA with spike-in controls using an Agilent One Color Spike Mix Kit (Agilent Technologies) was reverse transcribed into double strand cDNAs by AffinityScript multiple temperature reverse transcriptase and amplified for 2 h at 40°C. Resulting cDNAs were subsequently used for in vitro transcription by T7-polymerase and labeled with cyanine-3-labeled cytosine triphosphate (Perkin Elmer) for 2 h at 40°C using a Low Input Quick-Amp Labeling Kit ver. 6.5 (Agilent Technologies) according to the manufacturer's protocol.
|After labeling, rates of dye incorporation and quantification were measured with a Nanodrop ND-1000 spectrophotometer (Thermo Scientific) and were then fragmented for 30 min at 60°C in the dark. The labeled 1,650 ng of each cRNA sample was then hybridized on Agilent 4 × 44K whole mouse genome arrays (Agilent Design #026655) at 65°C for 17 h with rotation in the dark. Hybridization was performed using a Gene Expression Hybridization Kit (Agilent Technologies) following the manufacturer's instructions.
|After washing in GE washing buffer, slides were scanned with an Agilent Microarray Scanner G2505C.
Acute colitis model was induced to WT mice by addition of 1.75% DSS in drinking water for 7 days.
Pool from 3 mice.
|Feature Extraction software (Version 10.7.1.1) employing defaults for all parameters was used to convert images into gene expression data. Raw data were imported into GeneSpring GX 12.0 (Agilent Technologies) for database management and quality control. For normalization in GeneSpring, the threshold raw signal was set to 1.0 and the global normalization algorithm (percentile shift) was used with a percentile target of 75. This normalized data was used for identifying differentially expressed genes, in which KO group were compared to WT group as control.
|Jul 03, 2013
|Last update date
|Apr 17, 2014
|Immunology Frontier Research Center
|Human Immunology （Single Cell Genomics)
|Microarray analysis of colon tissue of DSS colitis STAP-2 KO mice