High resolution microarray c scanner

Fluorescently-labeled miRNAs were generated using the miRNA Complete Labeling and Hybridization kit (Agilent Technologies, Santa Clara, CA, USA), with a sample input of 100 ng of total RNA from BE63/3 and hybridized for 20 h at 55 °C on the Agilent 8 × 60 K Human miRNA Microarray slide (Agilent Technologies), based on miRBase database (Release 21.0). Following hybridization, the slides were washed and scanned using the High-Resolution Microarray C Scanner (Agilent Technologies). The image files were processed using the Agilent Feature Extraction software (v10.7.3): the microarray grid was correctly placed; inlier pixels were identified, and outlier pixels were rejected.

Microarray experiments were performed on biological triplicate samples. Microarray hybridizations were performed by the Transcriptomics and Genomics core facility of the Department of Emergency and Organ Transplants (DETO) – Nephrology Unit – of the University of Bari ‘Aldo Moro’ Italy¹. The labeled cRNA was produced using a Low Input Quick Amp Labeling (LIQA) kit (Agilent Technologies) and hybridized for 17 h at 65°C on an Agilent SurePrint G3 8 × 60K custom lncRNA expression array (Agilent Technologies). This array contains two probes for 22,001 lncRNAs targeting the Gencode v15 human lncRNA annotation, together with one probe for 17,535 randomly chosen protein-coding transcripts. After hybridization, the slide was washed according to Agilent protocols and scanned using a High-Resolution Microarray C Scanner (Agilent Technologies). The image file was processed using Agilent Feature Extraction software (v10.7.3). The microarray grid was correctly placed, and outlier pixels (which were rejected) and inlier pixels were identified. Normalization was performed according to the Quantile method. The differentially expressed probes were selected using a moderated t-test with a p-value cut-off of 0.05.