|
| Status |
Public on Mar 12, 2012 |
| Title |
wildtype_mock_3 |
| Sample type |
RNA |
| |
|
| Source name |
wildtype_mock treatment
|
| Organism |
Mus musculus |
| Characteristics |
strain background: BALB/c
genotype/variation: wild type
cell type: primary polarized airway epithelial cell cultures
|
| Treatment protocol |
mAEC cultures derived from balb/c WT, IFNAR-/- and STAT1-/- mice were exposed to 2 x 105 PFU influenza WSN for 2 h or mock inoculated and harvested 24 h post infection.
|
| Growth protocol |
Wild type balb/c mice were purchased from Harlan Laboratories, while balb/c IFNAR-/- mice and STAT1-/- mice were breed in house. All animals were maintained in BL2 containment under pathogen-free conditions. The Institutional Animal Care and Use Committee approved all the animal studies described in this work. Tracheal epithelial cell isolation and culture was performed as previously described (Rowe et al., 2004; You et al., 2002). Non-adherent cells were seeded onto 12mm 0.4 µM clear polyester membranes (Corning-Costar) previously coated with a collagen solution. After reaching confluence cells were incubated under ALI and when the transepithelial resistance was >1,000 W cm2 the basolateral media was replaced with fresh media every other day. Cultures were routinely used for experimentation 10-14 days post ALI.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from the AEC cultures after direct lysis of cells with TRIzol (Invitrogen) and application of the resulting aqueous phase to Qiagen RNeasy Mini Columns. 2 ug of total RNA was reversed transcribed in a 20 ul volume using Applied Biosystem’s high capacity reverse transcription Kit.
|
| Label |
Cy3
|
| Label protocol |
The concentration of the samples provided was determined using the NanoDrop® ND-1000 UV-Vis Spectrophotometer. RNA samples were analyzed by the FGC using an Agilent 2100 Bioanalyzer Lab-On-A-Chip Agilent 6000 Series II chip to determine the integrity of the samples. The RNA was of high quality and all samples passed our QC cutoff. Sample labeling and hybridization was performed according to the manufacturer’s protocols.
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| |
|
| Hybridization protocol |
Hybridization was performed following the manufacturer's protocols
|
| Scan protocol |
Microarray slides were hybridized overnight, washed and then scanned with Agilent G2505C Microarray Scanner. This high resolution scanner represents the very latest technology from Agilent for Arrays and features an industry-leading extended dynamic range of 106 (20-bits) for high sensitivity scanning without saturation, low-level detection resulting from optimized precision optics, broad dynamic range, minimal spectral cross talk that enables detection of weak features. The information about each probe on the array was extracted from the image data using Agilent Feature Extraction 10.5 (FE). This data is stored in the FE '.txt' files. The raw intensity values from these files is imported into the mathematical software package 'R', which is used for all data input, diagnostic plots, normalization and quality checking steps of the analysis process using scripts developed in-house by Peter White specifically for this analysis.
|
| Description |
gene expression after 2h mock treatment (harvested 24h after treatment)
|
| Data processing |
Preprocessing refers to the procedures used to convert raw probe intensities into useful gene expression measurements. For analysis of Agilent Expression data this process consists of : Reading in the FE extraction files and extraction the median intensity values for each probe on the array. These intensities are not background corrected (this has been shown to only introduce noise), but are corrected for any scanner offset that was added to the original raw values (see Analysis Log file for details). The dataset is filtered to remove positive control elements and any elements that have been flagged as outliers. Present (P), Marginal (M) or Absent (A) calls are made for each element on the array using in-house methodologies that take into account both the results of FE probe detection statistics and the intensities of the negative controls elements on the array. Data normalization is the process of removing unwanted non-biological variation that might exist between arrays in a microarray experiment. Sources of such variation might include scanner-setting differences, the quantities of mRNA hybridized, processing order and many other factors. Regardless of the normalization approach used, the underlying assumption of the normalization algorithm is that the number of genes changing expression between conditions is relatively small or that an equivalent number of genes increase and decrease in expression. Agilent One-Color Analysis: The median green (Cy3) intensities are normalized between the arrays using the Quantile Normalization package in 'R' (Bolstad et al., 2003). Quantile normalization is a non-linear probe-level normalization that results in the same empirical distribution of intensities for each array.This is a significantly more robust approach than simply normalizing to the median value of each array.The expression of each condition then was normalized to its respective control: wild type mAECs infected with WSN were normalized to wild type mock treated mAECs, IFNaR-/- mAECs infected with WSN were normalized to IFNaR-/- mock treated mAECs etc.
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| |
|
| Submission date |
Sep 15, 2011 |
| Last update date |
Mar 12, 2012 |
| Contact name |
IOANNIS IOANNIDIS |
| E-mail(s) |
[email protected]
|
| Phone |
614-355-3038
|
| Organization name |
NATIONWIDE CHILDRENS HOSPITAL
|
| Department |
VACCINES AND IMMUNITY
|
| Lab |
FLANO LAB
|
| Street address |
700 CHILDRENS DRIVE
|
| City |
COLUMBUS |
| State/province |
OH |
| ZIP/Postal code |
43220 |
| Country |
USA |
| |
|
| Platform ID |
GPL7202 |
| Series (2) |
| GSE32137 |
The response of murine primary airway epithelial cells to Influenza infection and the importance of Interferon type I signaling in this response [mAEC]. |
| GSE32140 |
The response of PBMCs and primary airway epithelial cells to Influenza and RSV virus |
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