Nasal epithelial cells obtained by bilateral nasal brushing from control patient not suffering from CF ands assumed to be expressing wild type CFTR gene.
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).