Total RNA, including miRNAs, was extracted from the olfactory epithelium cells by using “Monarch® Total RNA Miniprep kit” (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Briefly, this kit is a comprehensive solution for sample preservation, cell lysis, gDNA removal, and purification of total RNA from a wide variety of biological samples, including mammalian tissue. Total RNA was quantified by Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity, and the content of miRNAs (%) in each sample were assessed by capillary electrophoresis with the RNA 6000 Nano LabChip and the Small RNA Nano LabChip, respectively, using the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Only samples with RNA Integrity Number (R.I.N.) values higher than six were used for gene and miRNA expression analysis.
Label
Cy3
Label protocol
Total RNA (200 ng) was labeled with pCp Cy3, according to the Agilent’s protocol, and unincorporated dyes were removed with MicroBioSpin6 columns (BioRad, Hercules, CA, USA)
Hybridization protocol
Probes were hybridized at 55°C for 22 hours using the Agilent's hybridization oven that is suited for bubble-mixing and microarray hybridization processes.
Scan protocol
Slides were scanned on an Agilent microarray scanner (model G2565CA) at 100% and 10% sensitivity settings. Agilent Feature Extraction software version 12.0.0.7 was used for image analysis.
Description
miRNA expression profiling of patient with no olfactory symptoms (Group 3, P22)
Data processing
Inter-array normalization of miRNA expression levels was performed with cyclic Lowess for miR [Risso et al., 2009] and the average of replicates was used. Feature Extraction software (Agilent Technologies) was used to obtain spot quality measures in order to evaluate the quality and the reliability of the hybridization. In particular, the flag “glsFound” (set to 1 if the spot has an intensity value significantly different from the local background, 0 otherwise) was used to filter out unreliable probes: flag equal to 0 will be noted as “not available” (NA). So, in order to perform a more robust and unbiased statistical analysis, probes with a high proportion of NA values were removed from the dataset. NA (44%) was used as threshold in the filtering process, obtaining a total of 210 available human miRNAs.
