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Sample GSM2196361 Query DataSets for GSM2196361
Status Public on Feb 13, 2018
Title LAIV_B334_36h_1
Sample type RNA
 
Source name Nasal Epithelial Cells
Organism Homo sapiens
Characteristics tissue: Nasal Epithelial Cells
infection: LAIV
sampleID: B334
timepoint: 36h
Treatment protocol Differentiated hNECs where infected at a multiplicity of infection (MOI) of 5 50% tissue culture infectious dose per cell. All incubations were performed at 32oC in a humidified environment with 5% CO2. Prior to infection, cells were apically washed with 200 μl DMEM (supplemented with 0.3% BSA, 2mM Glutamax, 100 U/ml Penicillin, and 100 µg/ml Streptomycin) and the basolateral media was refreshed with 500 μl of fresh LHC Basal Medium:DMEM-H. The virus inoculum was added to the apical compartment in a volume of 100 μl and incubated for 2 hours, after which the inoculum was aspirated and the apical chamber was washed three times with 200 μl phosphate buffered saline containing calcium and magnesium (PBS+). The plates were then returned to the incubator. Both apical washes and basolateral medium were collected and stored at -70oC. Virus production each time point was quantified by limited dilution infection of MDCKs using apical wash samples. The TCID50 for each sample was calculated using the Reed-Muench algorithm (Reed, 1938). RNA was isolated from Trizol homogenates from samples harvested at 24hpi and 36hpi.
Growth protocol Human nasal epithelial cells (hNECs) were obtained from non-diseased hosts during endoscopic sinus surgery for non-infection related conditions and grown in culture at the air-liquid interface (ALI) as previously described (Lane et al., 2004; Ramanathan and Lane, 2007; Ramanathan et al., 2007). Tissue processing, differentiation medium, and culture conditions have been previously described in detail (Fischer et al., 2015). Seasonal influenza A virus A/Victoria/361/2011 (H3N2) and an antigenically-matched live attenuated influenza vaccine virus (LAIV; WT HA and NA, all other proteins from A/Ann Arbor/6/1960) were utilized in this study. Viral seed stocks were obtained from Medimmune.
Extracted molecule total RNA
Extraction protocol All Trizol lysates were processed simultaneously: they were phase-separated, and RNA was isolated from the aqueous phase (diluted 2 fold with RLT buffer) using Qiagen RNeasy Mini columns and the manufacturer’s recommended protocol (Qiagen Inc., Valencia, CA). RNA quality was assessed on an Agilent 2100 Bioanalyzer using the nanochip format, and only intact RNA was used for microarray analyses.
Label Cy3
Label protocol The Agilent One-Color Microarray-Based Gene Expression Analysis Protocol was followed for the Cy3-cDNA probe preparation, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.
 
Hybridization protocol The Agilent One-Color Microarray-Based Gene Expression Analysis Protocol was followed for hybridization and array washing. Two hundred fifty ng of each RNA sample was hybridized to one Agilent 4X44K mouse array.
Scan protocol Dry slides were scanned on an Agilent DNA microarray scanner (Model G2505B) using the XDR setting.
Data processing Raw images were analyzed using the Agilent Feature Extraction software (version 9.5.3.1) and the GE1-v5_95_Feb07 extraction protocol.
 
Submission date Jun 10, 2016
Last update date Feb 13, 2018
Contact name Michael Katze
E-mail(s) data@viromics.washington.edu
Organization name University of Washington
Department Microbiology
Lab Michael G. Katze, Ph.D
Street address Rosen Building 960 Republican St.
City Seattle
State/province WA
ZIP/Postal code 98109-4325
Country USA
 
Platform ID GPL17077
Series (2)
GSE83215 Evaluation of the immunogenicity of live-attenuated influenza vaccines in nasal epithelial cells in primary differentiated human nasal epithelial cells [Microarray Expression]
GSE83285 Evaluation of the immunogenicity of live-attenuated influenza vaccines in nasal epithelial cells in primary differentiated human nasal epithelial cells

Data table header descriptions
ID_REF
VALUE Log2 quantile-normalized signal intensity

Data table
ID_REF VALUE
A_23_P117082 11.76598989
A_33_P3246448 4.59662662
A_33_P3318220 4.47228916
A_33_P3236322 5.342259844
A_33_P3319925 5.444415343
A_21_P0000509 15.91057519
A_21_P0000744 10.52765293
A_24_P215804 6.777379525
A_23_P110167 13.7761216
A_33_P3211513 8.806317213
A_23_P103349 3.432201148
A_32_P61480 6.908391541
A_33_P3788124 3.946476633
A_33_P3414202 8.301650783
A_33_P3316686 9.492667768
A_33_P3300975 4.33606511
A_33_P3263061 13.41436582
A_33_P3261373 3.759173344
A_24_P278460 9.10030017
A_21_P0013109 4.197001323

Total number of rows: 50683

Table truncated, full table size 1258 Kbytes.




Supplementary file Size Download File type/resource
GSM2196361_US93503719_253949435318_S01_GE1_107_Sep09_2_2.txt.gz 12.3 Mb (ftp)(http) TXT
Processed data included within Sample table

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