2.2.1. RNA Extraction and Analysis

Temporal cortex samples were homogenised for 30 s using a TissueRuptor II (Qiagen, Hilden, Germany) in RNase-free PBS (ThermoFisher Scientific, Waltham, MA, USA). RNA was extracted from the homogenised samples using the miRNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The RNA concentration was measured on a nanophotometer (IMPLEN, Westlake Village, CA, USA).

An Agilent 2100 Bioanalyzer (Santa Clara, CA, USA) was used to detect the size distribution of total RNA as well as determine the quality of the RNA. A total of 100 ng of RNA was extracted and converted to cDNA using the miRCURY LNA kit (Qiagen, Hilden, Germany). cDNA was mixed with a SYBR master-mix (miRCURY SYBR Green Master Mix; Qiagen, Hilden, Germany). This was added to the miRCURY LNA miRNA miRNome Panel I–II using the automated pipettor the QIAgility (Qiagen, Hilden, Germany). These plates were run on the Quantstudio 5 qPCR machine (ThermoFisher Scientific, Waltham, MA, USA) as instructed by protocol provided with the panels. In total 751miRNAs (miRCURY LNA miRNA miRNome PCR Panels, https://geneglobe.qiagen.com/us/product-groups/mircury-lna-mirna-mirnome-pcr-panels, accessed on 6 October 2019 were tested using the miRNome plates for each of the samples. Overall, 705 miRNAs showed Cq values, and fold changes were calculated using the ΔΔCq method. hsa-miR-99a-5p and hsa-miR-361-5p were selected as the most stable pair of miRNAs across the samples (stability value = 0.05) by NormFinder software (https://moma.dk/normfinder-software, accessed on 1 December 2019 and therefore used as the housekeeping genes.