|
| Status |
Public on Jan 29, 2009 |
| Title |
Patient_526_Current_Smoker |
| Sample type |
RNA |
| |
|
| Source name |
epithelial cells from right mainstem bronchus
|
| Organism |
Homo sapiens |
| Characteristics |
Sex: Male
Race: Hispanic
Age: 25
PKYRS: 3
Current smoker
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Epithelial cells were obtained from brushing the right mainstem bronchus of current and never smoker volunteers at bronchoscopy. Epithelial cell content yields were generally approximately 90%. RNA was size fractionated, and low molecular weight RNA ( < 200 nucleotides) was obtained from each sample for microRNA microarray analysis.
|
| Label |
fluorescent dendrimer with approximately 15 molecules of Oyster®-550 dye
|
| Label protocol |
Three hundred nanograms 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.
Patient526.gpr
|
| 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/). Principle Component Analysis (PCA) was performed across all samples using all miRNAs to assess the relative homogeneity of the expression values between samples. Based on PCA, one non-smoker sample was removed from further analysis. In addition to our sample filter, only miRNA replicates that were detected at levels two standard deviations above the average background in at least 6 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, leaving a total of 232 distinct miRNAs that were analyzed further.
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| |
|
| Submission date |
Jan 29, 2009 |
| Last update date |
Jan 29, 2009 |
| Contact name |
Sriram Sridhar |
| E-mail(s) |
sridhars@medimmune.com
|
| Phone |
6178771485
|
| Organization name |
MedImmune
|
| Department |
Translational Medicine
|
| Street address |
1 MedImmune Way
|
| City |
Gaithersburg |
| State/province |
MD |
| ZIP/Postal code |
20878 |
| Country |
USA |
| |
|
| Platform ID |
GPL8131 |
| Series (2) |
| GSE14224 |
MicroRNAs as modulators of smoking-induced gene expression changes in human airway epithelium |
| GSE14634 |
MicroRNA expression from bronchial epithelial cell samples of current and never smokers. |
|
