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Status |
Public on Mar 18, 2019 |
Title |
BENaC_Sham_Female_1day_Rep5 |
Sample type |
RNA |
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Source name |
RNA isolated from whole lung tissue from female BENaC mice given 1 day of sham exposure
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Organism |
Mus musculus |
Characteristics |
tissue: Homogenized whole lung tissue genotype: Scnn1b-overexpressing (BENaC) exposure: Sham Sex: Female exposure duration: 1 day
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Treatment protocol |
Sex-matched 5-7-week old WT and βENaC littermates were exposed to cigarette smoke or sham (room air) exposure. Each exposure and genotype group included both males and females (n=5 animals of each sex per group for most groups. In the 5-day female group, there were n=6 WT sham animals and n=4 βENaC sham animals). Exposure occurred in a plexiglass chamber attached to a smoke delivery device using an exposure chamber and smoking machine (inExpose Exposure System, SCIREQ, Chandler, AZ). The chamber contained pie-slice separators and positions for 16 mice. Mice were exposed to mainstream + side-stream smoke from 6 reference cigarettes with filters removed per day (College of Agriculture Reference Cigarette Program, University of Kentucky, 3R4F research cigarettes) [18]. Each cigarette was puffed for 2 sec every 25 sec, using the standard Federal Trade Commission smoking machine protocol [18]. The sham-exposed control mice were exposed to room air in the exposure chamber for a time equivalent to that needed for active smoke exposure. Mice were exposed to cigarette or sham smoke for 1 day or 5 consecutive days. Samples were harvested 4 hours after the completion of the final smoke exposure. The right lung was used for gene expression analysis.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was isolated using miRNeasy kits from Qiagen (Valencia, CA, USA) according to the manufacturer's instructions. RNA was isolated from lung tissue homogenates using the miRNeasy kit (Qiagen). Spectrophotometric ratios of A260/A280 and A260/A230 were 1.7-2.1 and greater than 1.6, respectively. RIN values were greater than 7.4; the average RIN for all samples was 9.1.
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Label |
biotin
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Label protocol |
Total RNA (250 ng) was used to synthesize fragmented and labeled sense-strand cDNA and hybridize onto Affymetrx arrays. All 80 samples were run on the same 96 well array plate. The Affymetrix HT WT User Manual was followed to prepare the samples. Briefly, the WT Expression HT Kit for Robotics (Ambion) was used to generate sense-strand cDNA from total RNA. Following synthesis of sense-strand cDNA, the cDNA was fragmented and labeled with the Affymetrix GeneChip HT Terminal Labeling Kit. The Beckman Coulter Biomek FXP Laboratory Automation Workstation with the Target Express set up was used to prepare the samples with these two kits.
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Hybridization protocol |
Fragmented and labeled cDNA was used to prepare a hybridization cocktail with the Affymetrix GeneTitan Hybridization Wash and Stain Kit for WT Arrays. Hybridization, washing, staining and scanning of the Affymtrix peg plate arrays was carried out using the Affymetrin GeneTitan MC Instrument. GeneChip Command Console Software (AGCC) was used for GeneTitan Instrument control.
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Scan protocol |
Scanning of the Affymetrix peg plate arrays was carried out using the Affymetrix GeneTitan MC Instrument. GeneChip Command Console Software (AGCC) was used for GeneTitan Instrument control.
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Data processing |
Data were evaluated using Affymetrix Expression Console v1.4 software for quality control based on summary statistics, and Partek Genomics Suite v6.6 for normalization. Expression signals from CEL files were preprocessed and normalized by RMA (Robust Multiarray Average) background correction, GC content and probe sequence correction, quantile normalization, and median polish summarization of probe signals mapped to specific genes. Custom probeset-to-gene mappings were generated from Affymetrix Probeset and Transcript Annotation release 35 by consolidating all probesets mapped, in order of preference, to Ensembl 81 gene ID, Refseq mRNA, and Genbank accession numbers. The RMA-normalized log2 intensity values were used as input for the General Linear Model (glm) function in R.
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Submission date |
Jan 29, 2018 |
Last update date |
Mar 18, 2019 |
Contact name |
Claire M Doerschuk |
Organization name |
UNC Chapel Hill
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Street address |
125 Mason Farm Rd. CB#7248
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City |
Chapel Hill |
State/province |
NC |
ZIP/Postal code |
27599 |
Country |
USA |
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Platform ID |
GPL22070 |
Series (1) |
GSE109776 |
Gene expression after 1 and 5 days of cigarette smoke exposure in mice with chronically inflamed or healthy lungs |
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