|
| Status |
Public on Dec 31, 2013 |
| Title |
6982-11 |
| Sample type |
RNA |
| |
|
| Source name |
Whole lung tissue
|
| Organism |
Homo sapiens |
| Characteristics |
copd status: Donor
slice: 11
lm: 409.877279120464
Sex: Male
age: 59
pack years: Non-Smoker
notes: Hypertension
disease state: control
tissue: lung
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Low molecular weight RNA was isolated from tissue cores using the miRNeasy Mini Kit (Qiagen). The RNA integrity was assessed using an Agilent 2100 Bioanalyzer and RNA purity was assessed using a NanoDrop spectrophotometer. 300 ng of low molecular weight RNA was processed and used as starting material for the microarray studies
|
| Label |
fluorescent dendrimer with approximately 15 molecules of Oyster®-550 dye
|
| Label protocol |
300 ng of LMW RNA were labeled using the FlashTag labeling kit (Genisphere, Inc., Hatfield, PA). MicroRNA were first poly-A tailed and then directly ligated to a fluorescent dendrimer (a branched DNA structure carrying approximately 15 molecules of Oyster®-550 dye (DeNovo Biolabels GmBH, Munster, Germany)).
|
| |
|
| Hybridization protocol |
5uL of 10% BSA and 28.5uL of 2X enhanced hybridization buffer (Genisphere, Hatfield, PA) were added to the sample to make the hybridization mix. This was applied to Invitrogen NCode miRNA microarrays containing 1053 miRNAs from 6 species (467 human miRNAs) printed in triplicate. The arrays incubated overnight (18 hours) in a humidified chamber at 52oC and the following morning, serial 15 minute washes were done with 2x SSC/0.2% SDS at 52oC, 2x SSC at room temperature and 0.2X SSC at room temperature.
|
| Scan protocol |
After drying, the arrays were scanned on a GenePix 4000 scanner (Axon/Molecular Devices, Sunnyvale CA).
|
| Description |
Low molecular weight RNA was hybridized to Invitrogen NCode microRNA microarrays, containing 1053 microRNAs across 6 species, including 467 human microRNAs. All microRNA spots were printed in triplicate. Blank spots containing no printed sequence served as background. Data was quantile normalized and log transformed. Only miRNA replicates 2 standard deviations above background were considered for further analysis
|
| Data processing |
Raw data was extracted using GenePix Pro 4.0 (Molecular Devices Corporation, Sunnyvale, CA), quantile normalized and log transformed using the limma package for R (http://www.r-project.org/). Only miRNA replicates that were detected at levels two standard deviations above the average background in at least 80% of samples were considered for further analysis. The median of the remaining replicates for each miRNA was then taken to obtain a single expression value for each miRNA.
|
| |
|
| Submission date |
Aug 14, 2013 |
| Last update date |
Dec 31, 2013 |
| Contact name |
Stephanie A Christenson |
| Organization name |
University of California, San Francisco
|
| Department |
Pulmonary and Critical Care
|
| Street address |
513 PARNASSUS AVENUE, HSE1305
|
| City |
San Francisco |
| State/province |
California |
| ZIP/Postal code |
94143 |
| Country |
USA |
| |
|
| Platform ID |
GPL15892 |
| Series (1) |
| GSE49881 |
microRNA expression associated with emphysematous lung destruction |
|
