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Sample GSM994337 Query DataSets for GSM994337
Status Public on Apr 10, 2013
Title CF nasal epithelium_biological rep5
Sample type RNA
 
Source name Nasal epithelial cells obtained by bilateral nasal brushing from CF patient with confirmed F508del homozygosity for the CFTR gene.
Organism Homo sapiens
Characteristics disease: cystic fibrosis
age: 10
gender: F
Treatment protocol Following washing and centrifugation in PBS, the aliquot of cells destined for RNA extraction was suspended in RLT extraction buffer from the Qiagen RNeasy kit on ice, and RNA extracted immediately upon return to the laboratory, as described below.
Growth protocol The study was conducted at the Faculty of Sciences of the University of Lisboa with samples collected at the CF Clinic of the Department of Pediatrics of the Santa Maria Hospital of Lisboa, and was approved by the Santa Maria Hospital Ethical Review Board. Informed consent was obtained from each participant, or parent/tutor where the participant was a minor. To be eligible for the study, individuals with CF had been previously confirmed to be homozygous for the F508del mutation. Individuals with recent viral infection, or active CF exacerbation were excluded. Non-CF control subjects were recruited from healthy volunteers attending the paediatric clinic for unrelated reasons. If participants demonstrated obvious turbinate inflammation or haemorrhage on initial brushing, the sample was excluded. Bilateral nasal mucosal brushing to collect respiratory epithelium was performed on each subject. An aliquot was set aside for cytological evaluation, and the remainder used for RNA extraction. A cytospin centrifuge (Shandon, Thermo Scientific, USA) was used to prepare formaldehyde-fixed respiratory epithelial samples for standard May-Grünwald-Giemsa (MGG) staining. Identity of samples was obscured with randomly numbered labels for blind counting. Slides were then evaluated twice using light microscopy and digital photography of 5 high power fields of view per sample, and cells were categorized as epithelial or inflammatory. Samples with more than 10% inflammatory cells were excluded from the study.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Concentration and purity was determined by spectrophotometry (Nanodrop) and integrity (RIN>7.0: mean RIN, CF - 8.2; Control - 8.4) was confirmed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay (Agilent Technologies, Palo Alto, CA).
Label biotin
Label protocol RNA was processed for use on Affymetrix (Santa Clara, CA, USA) GeneChip HsAirwaya520108F Arrays according to the manufacturer’s Two-Cycle Target Labelling Assay. Briefly, 90 ng of total RNA containing spiked in Poly-A RNA controls (GeneChip Expression GeneChip Eukaryotic Poly-A RNA Control Kit; Affymetrix) were used in a reverse transcription reaction (Two-Cycle DNA synthesis kit; Affymetrix) to generate first-strand cDNA. After second-strand synthesis, double-stranded cDNA was used in an in vitro transcription (IVT) reaction to generate cRNA (MEGAscript T7 kit; Ambion, Austin, TX). 600 ng of the cRNA obtained was used for a second round of cDNA and cRNA synthesis, resulting in biotinylated cRNA (GeneChip Expression 3’-Amplification Reagents for IVT-Labeling; Affymetrix). Size distribution of the cRNA and fragmented cRNA, respectively, were assessed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay.
 
Hybridization protocol 15 µg of fragmented cRNA was used in a 300-µl hybridization containing added hybridization controls. A final volume of 200µl was hybridized on arrays for 16 h at 45°C. Standard post hybridization wash and double-stain protocols (EukGE-WS2v5) were used on an Affymetrix GeneChip Fluidics Station 400.
Scan protocol Arrays were scanned on an Affymetrix GeneChip scanner 3000.
Data processing The data were normalized and summarized with RMA Express using default settings (Background Adjust: Yes, Normalization: Quantile).
 
Submission date Aug 29, 2012
Last update date Apr 10, 2013
Contact name Luka Clarke
E-mail(s) laclarke@fc.ul.pt
Organization name University of Lisboa
Department BioFIG
Street address Campo Grande
City Lisboa
ZIP/Postal code 1749-016
Country Portugal
 
Platform ID GPL10097
Series (1)
GSE40445 Gene expression in CF-vs-non CF human airway epithelial cell samples obtained by nasal brushing.

Data table header descriptions
ID_REF
VALUE log2 GC-RMA signal

Data table
ID_REF VALUE
1294_at 7.74088
1552257_a_at 8.426034
1552258_at 4.967728
1552261_at 8.20998
1552266_at 5.137603
1552269_at 6.059561
1552272_a_at 4.968876
1552281_at 6.932216
1552283_s_at 6.26186
1552286_at 6.354689
1552291_at 7.579221
1552296_at 5.539858
1552299_at 6.887444
1552301_a_at 4.465727
1552310_at 8.585638
1552312_a_at 6.202502
1552318_at 5.594393
1552326_a_at 6.960847
1552327_at 3.753035
1552330_at 6.055349

Total number of rows: 23064

Table truncated, full table size 450 Kbytes.




Supplementary file Size Download File type/resource
GSM994337_PM_CF_HsAirway.CEL.gz 1.9 Mb (ftp)(http) CEL
Processed data included within Sample table

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