|
| Status |
Public on Dec 22, 2011 |
| Title |
liver of control db/+ mice |
| Sample type |
RNA |
| |
|
| Source name |
Liver
|
| Organism |
Mus musculus |
| Characteristics |
tissue: liver
strain: db/+
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen liver tissues were minced and homogenized and total RNA was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA was quantified using Nanodrop spectrophotometer and RNA quality and integrity was analyzed in Bioanalyser.
|
| Label |
pCp-Cy3
|
| Label protocol |
miRNA labeling was performed as described in Agilent miRNA microarray protocol version 2.0 using Agilent miRNA labeling kit. 100 ng of total RNA was dephosphorylated with calf intestine alkaline phosphatase for 30min at 37°C. Denaturation was performed by adding DMSO and incubating at 100°C for 7 min and immediately transferred to ice water bath. Ligation was performed with pCp-Cy3 at 16 °C for 2 h. The labeled samples were dried completely in a vaccum concentrator and resuspended in 18 ul of nuclease free water.
|
| |
|
| Hybridization protocol |
The hybridization mixture [10X GE blocking agent (4.5 ul), 2X Hi-RPM hybridization buffer (22.5ul)] along with labelled miRNA sample was heated for 5 min at 100 °C and immediately cooled to 0 °C. Each 45 mL sample was hybridized onto a microarray at 55 °C for 20 h. Slides were washed 5 min in GE wash buffer 1 at RT and again for 5 min in GE wash buffer 2 at 37 °C, followed by an acetonitrile wash for 1 min at RT to dry the slides completely.
|
| Scan protocol |
Slides were scanned on an Agilent microarray scanner (model G2565BA) at 100 and 5% XDR settings. Agilent Feature Extraction software version 9.3.5 was used to extract the raw data.
|
| Description |
db/db mice are an excellent model of obesity and type 2 diabetes and exhibit insulin resistance as early as 10–12 days of age and at 8–12 wk, they exhibit glucose intolerance in response to an oral glucose challenge and a reduced hypoglycemic response to a bolus injection of insulin compared with nondiabetic control db/+ mice.
The mouse miRNA Microarray (V1) microarrays (G4472A) from Agilent Technologies was used for the experiment. It consisted of probes for 566 mouse miRNAs and 10 mouse viral miRNA.
|
| Data processing |
The raw data were global median normalized and log transformed and analyzed by Significance Analysis of Microarray (SAM). SAM calculates a score for each gene as a change of expression relative to the standard deviation of all measurements and therefore identifies genes that are significantly associated with an outcome variable such as the disease stage. In SAM tests, a false Discovery Rate (FDR) of less than 5% was selected and parameters were set as default.
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| |
|
| Submission date |
Jul 10, 2009 |
| Last update date |
Dec 22, 2011 |
| Contact name |
Malabika Datta |
| E-mail(s) |
mdatta@igib.res.in
|
| Phone |
91 11 27667602
|
| Organization name |
Institute of Genomics and Integrative Biology
|
| Street address |
Mall Road
|
| City |
Delhi |
| ZIP/Postal code |
110 007 |
| Country |
India |
| |
|
| Platform ID |
GPL7732 |
| Series (1) |
| GSE17035 |
microRNA microarray profiling in the livers of control db/+ and diabetic db/db mice |
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