miRNA Expression Profiling by Affymetrix Microarray

The miRNA expression profiling was carried out using Affymetrix platform-based microarrays with GeneChip™ miRNA 4.1 Array Strip (ThermoFisher Scientific, Waltham, MA, USA). The technical procedure that was followed was previously described in detail elsewhere [110 (link),111 (link)]. Each microarray was designed following the miRBase Release 20 database, which included complementary probes for 2578 human mature miRNA, 1996 human snoRNA, CDBox RNA, H/ACA Box RNA, scaRNA, and 2025 human pre-miRNA. miRNA was prepared for the hybridization step with the FlashTagTM Biotin HSR RNA Labeling Kit (ThermoFisher Scientific, Waltham, MA, USA). During this process, 150 ng of miRNA was subjected to the poly(A) tailing and biotin ligation in accordance with the producer’s protocol. Biotin-labeled miRNA were then hybridized to GeneChip™ miRNA 4.1 Array Strip (20 h, 48 °C). Afterwards, the microarrays were washed and stained following the technical protocol and using the Affymetrix GeneAtlas Fluidics Station (Affymetrix, Santa Clara, CA, USA). Next, the array strips were scanned using GeneAtlas System Imaging Station (Thermo Fisher Scientific, Waltham, MA, USA).