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Status |
Public on Sep 19, 2014 |
Title |
TEC-Media control biological replicate 4 technical replicate 2_Cy3 |
Sample type |
RNA |
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Source name |
Tracheal epithelial cell
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Organism |
Gallus gallus |
Characteristics |
gender: Female age: 5 weeks tissue: Trachea
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Treatment protocol |
TECs were exposed to either 500 MOI live Mycoplasma gallisepticum Rlow or Rhigh or 5µg/mL LAMPs from either strain for 1.5 hours for microarray experiments.
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Growth protocol |
Chicken tracheal epithelial cells were isolated from chicken trachea and cultured in ATE medium (described in manuscript) at 37C with 5% CO2 in a cell culture incubator for 96 hours. Mycoplasma gallisepticum strains were grown in Hayflicks media at 37C. All LAMPs were isolated using TX-114 phase seperation.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted using a combination protocol of Trizol-Chloroform extraction followed by purification using Qiagen RNEasy Mini kit (Qiagen, Valencia, CA)
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Label |
Cy3
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Label protocol |
Cyanine-3 (Cy3) and Cyanine-5 (Cy5) labeled cRNA was prepared from 50 ng RNA using the Two-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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Hybridization protocol |
1.5 ug of total cRNA (750 ng Cy3-labelled cRNA and 750 ng Cy-5-labelled) (specific activity >7 pmol Cy3/ug cRNA or >7 pmol Cy5/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
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Scan protocol |
Slides were scanned immediately after washing on the GenePix 4000B Laser Scanner using two color scan setting (Scan resolution 5um). PMT gains were adjusted to give equal ratio of Cy3 and Cy5 intensity while keeping saturated features to less than 1%.
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Description |
R(low)3-4.gpr Gene expression upon exposure to media control for 1.5 hr
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Data processing |
The scanned images were analyzed with GenePix Pro 7 for feature extraction (Molecular Devices) using default parameters to obtain background subtracted Signal intensities for each channel. Background intensity was calculated as the mean value of the 5 closest negative control features. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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Submission date |
Sep 18, 2014 |
Last update date |
Sep 19, 2014 |
Contact name |
Frank Zappulla |
E-mail(s) |
frank.zappulla@engr.uconn.edu
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Organization name |
University of Connecticut
|
Department |
Pathobiology
|
Street address |
61 North Eagleville Road
|
City |
Storrs |
State/province |
CT |
ZIP/Postal code |
06269 |
Country |
USA |
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Platform ID |
GPL15357 |
Series (1) |
GSE61520 |
Mycoplasma gallisepticum Lipid Associated Membrane Proteins Up-regulate Inflammatory Genes in Chicken Tracheal Epithelial Cells via TLR-2 Ligation Through an NF-κB Dependent Pathway |
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