|
| Status |
Public on Mar 15, 2011 |
| Title |
Colon_Normal_Adult_4 |
| Sample type |
RNA |
| |
|
| Source name |
Normal Colon
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: Normal Colon
genotype: TCF4WT ApcHet
|
| Growth protocol |
For gene expression in tumors, RNA was isolated from the distal 2.5 cm of colon collected from four each of Tcf4HetApcMin and Tcf4WTApcMin littermates.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using an RNAqueous RNA isolation kit (Ambion, Foster City, CA).
|
| Label |
Cy3
|
| Label protocol |
The Agilent One-Color Quick Amp Labeling Kit is used to generate fluorescently labeled cRNA for one-color microarray hybridizations. Agilent RNA spike-in controls are combined with input total RNA samples (50 to 1000 ng). The polyadenylated fraction of the total RNA sample is primed with oligo dT/T7 RNA polymerase promoter oligonucleotide sequences and cDNA synthesis is accomplished through the addition of MMLV-RT. Following cDNA synthesis, T7 RNA polymerase and cyanine 3-CTP nucleotides are combined with the reaction mixture to simultaneously amplify the target material through the generation of cRNA and incorporate cyanine 3-CTP. Fluorescently labeled, cRNA molecules are purified from the reaction mixture using the Qiagen RNeasy mini kit. The concentration of the purified samples is determined using a NanoDrop ND-1000 spectrophotometer.
|
| |
|
| Hybridization protocol |
Fluorescently labeled cRNA samples (825 ng each) were fragmented and combined with Agilent Hi-RPM Hybridization Buffer. Microarray hybridizations were performed using Agilent SureHyb Hybridization chambers. Hybridization chambers were loaded onto a rotisserie in an Agilent Hybridization oven and were incubated at 65Ã,ºC for 17 hours with a rotational speed of 10 rpm. Following incubation, the microarray slide was washed for 1 minute each in Gene Expression Wash Buffer 1 (6X SSPE, 0.005% N-lauroylsarcosine; room temperature) and Gene Expression Wash Buffer (0.06X SSPE, 0.005% N-lauroylsarcosine; room temperature) for 1 minute each. Microarray slides were briefly dipped in a solution of acetonitrile and dried.
|
| Scan protocol |
Microarray slides were scanned in an Agilent Technologies G2505C Microarray Scanner at 5 um resolution. The scanner performs simultaneous detection of Cyanine-3 and Cyanine-5 signal on the hybridized slide. Data captured from the scanned microarray image is saved as a TIF file.
|
| Description |
6398E7_Normal
|
| Data processing |
TIF files generated from the scanned microarray image are loaded into Agilent Feature Extraction Software version 10.1.1.1. The software automatically positions a grid and finds the centroid positions of each feature on the microarray. This information is used to perform calculations that include feature intensities, background measurements and statistical analyses. Data generated by the software is recorded as a tab-delimited text file.
Intensity data from Agilent one-color gene expression arrays was log-transformed (to log base 2) and quantile normalized. Data from microarray features flagged as non-uniform by the Agilent Feature Extraction software were removed prior to normalization.
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| |
|
| Submission date |
Feb 25, 2011 |
| Last update date |
Mar 15, 2011 |
| Contact name |
Mario R. Capecchi |
| Organization name |
University of Utah
|
| Street address |
15 North 2030 East
|
| City |
Salt Lake City |
| State/province |
UT |
| ZIP/Postal code |
84112 |
| Country |
USA |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE27522 |
Gene expression in Tcf4Het ApcHet mouse colon tumors compared to normal colon |
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