Gene Expression Analysis in ASD Model

RNA was isolated following the manufacturer’s protocol using the RNA Mini Kit (A&A Biotechnology, Gdańsk, Poland). The total RNA concentration was measured using an ND-1000 Spectrometer (NanoDrop Technologies Inc., Wilmington, DE, USA). One microgram of total RNA was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA).
In this study, we used 384-well TaqMan Gene Expression Custom Array Cards 24 (Cat# 4342249, Applied Biosystems), designed to cover different gene families relevant to ASD, selected based on the literature, the SFARI database, and previous RNA-seq analyses from the frontal cortex in the same model (PRJNA669556 BioProject) [30 (link)]. The gene set listed in Supplementary Table S1 and housekeeping genes (18S rRNA and Hprt1) were studied. Real-time (RT)-qPCR was carried out using Life Technologies TaqMan reagents (e.g., TaqMan Fast Advanced Master Mix) according to the manufacturer’s protocol using a QuantStudio 12K Flex Real-Time PCR System (Applied Biosystems). Data were further analyzed with QuantStudio 12K Flex Expression Suite Software (Applied Biosystems). Quantification cycle data were normalized to 18S rRNA. The relative gene expression was calculated using the 2^−∆∆Ct (fold change) method.