|
| Status |
Public on Nov 01, 2010 |
| Title |
IGF-1 stimulated mouse small intestinal crypt cell vs control mouse small intestinal crypt cell replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
mouse small intestinal crypt cells
|
| Organism |
Mus musculus |
| Characteristics |
passage: 1
cell type: crypt cells isolated from mouse small intestine
strain: C57BL
|
| Treatment protocol |
untreated
|
| Growth protocol |
The cells were resuspended with crypt culture media (Advanced DMEM/F12, GlutaMax 1:100, Hepes 10 mM, Penicillin/Streptomycin 1:100, N2 supplement 1:100, B27 supplement, retinoic acid free 1:50, mouse recombinant EGF 50 ng/ml), mouse recombinant noggin 100 ng/ml, human recombinant R-spondin1 500 ng/ml, and N-Acetylcysteine 1 μM).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted with miRNeasy Mini Kit (Qiangen) according to the manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
300 ng of each sample was labeled with Cy5/Cy3 using miRCURY LNA microRNA array Power Labeling kit (Exiqon).
|
| |
|
| Channel 2 |
| Source name |
mouse small intestinal crypt cells treated with 20ng/ml IGF-1
|
| Organism |
Mus musculus |
| Characteristics |
passage: 1
cell type: crypt cells isolated from mouse small intestine
strain: C57BL
|
| Treatment protocol |
20ng/ml IGF1 (R&D Systems)
|
| Growth protocol |
The cells were resuspended with crypt culture media (Advanced DMEM/F12, GlutaMax 1:100, Hepes 10 mM, Penicillin/Streptomycin 1:100, N2 supplement 1:100, B27 supplement, retinoic acid free 1:50, mouse recombinant EGF 50 ng/ml), mouse recombinant noggin 100 ng/ml, human recombinant R-spondin1 500 ng/ml, and N-Acetylcysteine 1 μM). IGF-1 was added after cells were plated onto the cell culture plate.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted with miRNeasy Mini Kit (Qiangen) according to the manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
300 ng of each sample was labeled with Cy5/Cy3 using miRCURY LNA microRNA array Power Labeling kit (Exiqon).
|
| |
|
| |
| Hybridization protocol |
Hybridization SureHyb chamber kit and Gasket slide kit (Agilent) were used to hybridize the labeled RNA for 18 h to Exiqon miRCURY LNA miRNA array V.11.0 (miRBase Sequence Database, http://microrna.sanger.ac.uk/sequences, release 11.0).
|
| Scan protocol |
Arrays were scanned on an Axon GenePix 4000B scanner
|
| Description |
n/a
|
| Data processing |
GPR files containing fluorescent ratios (sample/control) were generated using GenePix Pro 6.0 software. GPR files were read into R/Bioconductor [19] using the marray package. GenePix flagged spots were removed from subsequent analysis, and only unflagged human, mouse and rat probes were used for normalization and subsequent analysis. M (log2 ratios) of Cy5 to Cy3 signals were calculated for each array, and normalized by print tip loess normalization using only unflagged spots.
For each miRNA with more than 1 unflagged probe, the median of normalized M of the replicate probes was taken as its summary value for the miRNA in each array.
|
| |
|
| Submission date |
Feb 01, 2010 |
| Last update date |
Sep 27, 2010 |
| Contact name |
Bo Lonnerdal |
| E-mail(s) |
bllonnerdal@ucdavis.edu
|
| Organization name |
University of California, Davis
|
| Department |
Nutrition
|
| Lab |
3329 Meyer Hall
|
| Street address |
1 Shields Ave.
|
| City |
Davis |
| State/province |
CA |
| ZIP/Postal code |
95616 |
| Country |
USA |
| |
|
| Platform ID |
GPL7723 |
| Series (1) |
| GSE20133 |
Murine small intestinal crypt cells: IGF-1 stimulated vs. control |
|
