|
| Status |
Public on Oct 02, 2010 |
| Title |
Kras_mut_3 |
| Sample type |
RNA |
| |
|
| Source name |
colorectal tissue
|
| Organism |
Mus musculus |
| Characteristics |
tissue: colorectal tissue
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation was carried out using the Qiagen RNeasy Mini Kit for animal tissues (Qiagen, Valencia, CA). The RNA quality and quantity was checked using an Agilent 2100 bio-analyzer and RNA 6000 nano-chips. Total RNA was used to generate biotin labeled cRNA using the Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX).
|
| Label |
Cy3
|
| Label protocol |
0.5 µg of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in vitro transcription reaction where single-stranded RNA (cRNA) was generated and labeled by incorporation of biotin-16-UTP.
|
| |
|
| Hybridization protocol |
A total of 0.75 µg of biotin-labeled cRNA was hybridised at 58C for 16 h to Illumina’s Sentrix MouseRef-6 Expression Bead-Chips (Illumina, San Diego, CA). The arrays were washed, blocked and the labelled cRNA was detected by staining with streptavidin-Cy3.
|
| Scan protocol |
The arrays were scanned using an Illumina BeadStation 500× Genetic Analysis Systems scanner and the image data extracted using the Illumina BeadStudio software, Version 3.0.
|
| Description |
replicate 3
M-RMG_EGFP_Cre-DMH_3
|
| Data processing |
Data files generated from Illumina BeadStudio software were imported into Genespring GX version 10.0 (Agilent, Santa Clara, CA, USA). The raw signals were thresholded to 1.0 and normalisation was carried out using the percentile shift algorithm and this was followed by baseline transformation to the median of all the samples. Hierarchical clustering was carried out with in house clustering software using the Pearson uncentered similarity measure and Ward linkage algorithm. Pathway analysis of the up and down regulated genes between the Kras mutants and controls were carried out using Ingenuity Pathway analysis (Ingenuity Systems, CA, USA).
|
| |
|
| Submission date |
Sep 09, 2010 |
| Last update date |
Oct 01, 2010 |
| Contact name |
Mark J Arends |
| E-mail(s) |
mja40@cam.ac.uk
|
| Phone |
01223217813
|
| Organization name |
University of Cambridge
|
| Department |
Pathology
|
| Street address |
Hills Road
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 0QQ |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL6481 |
| Series (1) |
| GSE24010 |
Mutant K-ras promotes carcinogen-induced murine colorectal tumourigenesis, but does not alter tumour chromosome stability |
|
