|
| Status |
Public on Mar 22, 2013 |
| Title |
9000149 |
| Sample type |
RNA |
| |
|
| Source name |
bronchial brushing
|
| Organism |
Homo sapiens |
| Characteristics |
patient number: 9705284
used in analysis: no
fev1%: NA
fev1/fvc: NA
copd: NA
age (years): NA
smoking status: NA
Sex: NA
pack years: NA
history of asthma: no
inhaled medications: no
tissue: bronchial epithelial cells
change in fev1: NA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Isolation of high molecular weight RNA was performed using the miRNeasy mini kit (Qiagen) as per the manufacturer's instructions. Briefly, bronchial epithelial cells were lysed and homogenized in QIAzol Lysis Reagent. After chloroform extraction, the aqueous phase containing RNA was mixed with ethanol. This was applied to an RNeasy Mini Spin Column to retain high molecular weight RNA (>200 nt). After washing, high molecular weight RNA was eluted.
|
| Label |
biotin
|
| Label protocol |
High molecular weight RNA was processed and hybridized to Affymetrix Human Gene Arrays ST v1.0 as described in the GeneChip® Whole Transcript (WT) Sense Target Labeling Assay Manual (Affymetrix). Briefly, 200ng of high molecular weight RNA was reverse transcribed (Whole Transcript cDNA Synthesis Kit, Affymetrix). The resulting cDNA was used as a template for in vitro transcription (IVT) (Whole Transcript cDNA Amplification Kit, Affymetrix). The resulting antisense cRNA was purified (GeneChip Sample Cleanup Module, Affymetrix) and then reverse transcribed with dUTP incorporation (Whole Transcript cDNA Synthesis Kit, Affymetrix, Santa Clara, CA). The resulting single-stranded sense DNA was fragmented using uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE1). The fragmented DNA was labeled using DNA Labeling Reagent which is covalently linked to biotin with terminal deoxynucleotidyl transferase (TdT) (Whole Transcript Terminal Labeling Kit, Affymetrix).
|
| |
|
| Hybridization protocol |
The labeled fragmented cDNA was hybridized to Affymetrix Human Gene ST v1.0 microarrays for 16-18 hours in the GeneChip Hybridization Oven 640 (45C, 60rpm rotation). The hybridized samples were washed. Using Affymetrix Fluidics Station 450 (Hybridization Washing and Staining Kit, Affymetrix), the samples were first stained with streptavidin (SAPE). This was followed by signal amplification with a biotinylated goat anti-streptavidin antibody and a second SAPE stain.
|
| Scan protocol |
Affymetrix GeneArray Scanner 3000 7G Plus (Affymetrix).
|
| Description |
AS_SL_E5_149
High MW RNA.
Gene expression data from bronchial brushing.
|
| Data processing |
RMA, EntrezGene v11.0.1 CDF.
|
| |
|
| Submission date |
Apr 10, 2012 |
| Last update date |
Apr 21, 2021 |
| Contact name |
Katrina Steiling |
| Organization name |
Boston Univeristy School of Medicine
|
| Department |
Medicine
|
| Lab |
Division of Computational Biomedicine
|
| Street address |
72 East Concord Street, E-631
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02118 |
| Country |
USA |
| |
|
| Platform ID |
GPL13243 |
| Series (1) |
| GSE37147 |
Bronchial airway gene expression reflects a COPD-associated field of injury that changes with disease severity and is reversible with therapy |
|
