individual: healthy subject age (years): 79 gender: M previous treatment: No atopy: No cell type: BSC cells isolated from: NM protocol: ALI culture time: day 14
Treatment protocol
Human primary basal stem/progenitor cells were not treated during ALI culture. Mucociliary differentiation was only induced by differentiation media (B-ALI (Lonza) supplemented with SingleQuots™ kit containing: BPE, hydrocortisone, hEGF, epinephrine, insulin, triiodothyronine, transferrin, GA-1000, inducer, and retinoic acid) and exposure to air.
Growth protocol
Human primary basal cells were obtained from CRSwNP patients (n=7) and healthy subjects (NM, n=7) by the explant technique in BEGM™ (LONZA) media suplemented with SingleQuots™ (LONZA) containing: BPE, hydrocortisone, hEGF, epinephrine, insulin, triiodothyronine, transferrin, GA-1000, and retinoic acid. Cells were cultured and differentiated at the air-liquid interface (ALI) system for 28 days in differentiation media composed of B-ALI™ (Lonza) supplemented with suplemented with SingleQuots™ (LONZA) containing: BPE, hydrocortisone, hEGF, epinephrine, insulin, triiodothyronine, transferrin, GA-1000, and retinoic acid.
Extracted molecule
total RNA
Extraction protocol
Total RNA, including small RNAs, was isolated from cells using the miRNeasy Mini Kit from Qiagen (Valencia, CA, USA). Briefly, cells were lysed with Qiazol lysis buffer (Qiagen), with cells from three inserts pooled for each time point, followed by passage through a Qiashredder column (Qiagen). Total RNA was extracted via RNeasy mini spin columns. All incubation and washing steps were performed following the manufacturer’s instructions.
Label
biotin
Label protocol
Total RNA (100 ng, RIN ≥ 9) was processed for global transcriptional analysis using the GeneChip® 3’ IVT Express kit and the Human Genome U219 24-Array Plate (Affymetrix, Santa Clara, CA, USA), following the manufacturer’s directions.
Hybridization protocol
Global transcriptional analysis using the GeneChip® 3’ IVT Express kit and the Human Genome U219 24-Array Plate (Affymetrix, Santa Clara, CA, USA), following the manufacturer’s directions.
Scan protocol
GeneTitan Affymetrix genetic analysis system
Description
Gene expression data from CONTROL NM ALI Cultures at day 14
Data processing
Microarray data (Affymetrix .cel files) were processed for raw data quality control analysis by using Robust Multichip Analysis (RMA) algorithm, which perform background correction, probe-level normalization, and probe-set summary.
Submission date
Dec 02, 2017
Last update date
Dec 01, 2018
Contact name
Borja Callejas-Díaz
Organization name
IDIBAPS, HOSPITAL CLÍNIC
Department
Clinical and Experimental Respiratory Immunoallergy
