Nd 1000 spectrometer

RNA was isolated with the TRIzol reagent (cat #15596026, ThermoFischer, Waltham, MA, USA) following the manufacturer’s protocol. The total RNA concentration was measured using an ND-1000 Spectrometer (NanoDrop Technologies Inc., Wilmington, DE, USA). The quality of RNA was determined by using an RNA 6000 Nano Lab-Chip Kit and an Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Based on the RNA integrity number (RIN > 7.5) values, 68 samples were selected for sequencing.

Total RNA is isolated from brain tissue using the mirVana^TM miRNA Isolation Kit (Thermo Fisher Scientific, MA, USA) that allows isolation of total RNA with excellent yields. Experiments are performed according to manufacturer protocols followed by RNA cleanup using RNeasy® Plus Mini Kit (Qiagen, Germany). RNA is stored at −80°C. RNA concentrations and quality is assessed by analyzing A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios with a ND-1,000 spectrometer (Nanodrop Technologies LLC, DE, USA).

The muscle biopsies specimens frozen immediately in liquid nitrogen were used for measuring abundance levels of selected transcripts. Frozen tissue samples were homogenized in 1 mL Trizol reagent (Invitrogen, Carlsbad, CA, USA) and total RNA was extracted according to the manufacturer protocol. RNA concentration was measured using a ND-1000 Spectrometer (NanoDrop Technologies Inc., Montchanin, DE, USA). Reverse transcription was performed with Omniscript Reverse Transcriptase enzyme (Qiagen Inc., Valencia, CA, USA) at 37°C for 60 minutes. The qPCR reactions were performed using Assay-On-Demand TaqMan probes (VEGFA, Hs00900055_m1; FLT1, Hs01052961_m1; KDR, Hs 00911700_m1; HIF1A, Hs00153153_m1; MFN1, Hs00966851_m1; MFN2, Hs00208382_m1, OPA1, Hs01047018_m1) according to the manufacturer's protocol (Applied Biosystems, Foster City, CA, USA) and were run on the CFX96 Real-Time system (Bio-Rad, Foster City, CA, USA). Expression of the hypoxanthine-guanine phosphoribosyltransferase (HPRT1, Hs01003270_g1) transcript with stable levels following experimental conditions was quantified to control for variation in cDNA amounts. The abundance of RNA was calculated as 2^{-(threshold cycle)}. Data were normalized to the expression levels of the HPRT1 mRNA.

Total RNA was prepared from snap-frozen hypothalamic and adrenal tissue using Qiagen RNeasy Lipid Tissue Mini Kit Cat # 74804 (Qiagen, CA, USA) according to the manufacturer’s instructions, and stored at -80 ^o C. RNA integrity was measured using a 2100 Bioanalyzer instrument and an RNA 6000 Nano LabChip assay (Agilent Technologies, CA, USA). RNA concentrations were determined by absorption at 260-nm wavelength with an ND-1000 spectrometer (Nanodrop Technologies, DE, USA).

Extraction of unique mRNA fingerprints was performed using RNeasy® Plus Mini Kit (Qiagen). RNA samples were stored at −80°C. Their quality and yield were assessed using A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ and ratios were analyzed with Experion™ Automated Electrophoresis System (Biorad). RNA concentrations were determined by absorption at 260 nm wavelength with a ND-1000 spectrometer (Nanodrop Technologies).
Gene expression profiling was performed using the GeneChip Human Genome U133 Plus 2.0 arrays (Affymetrix Inc.). 100 ng of RNA was amplified, labeled, fragmented, and hybridized using GeneChip 3′ IVT Express Kit. Washing and staining were conducted using GeneChip Fluidics Station 450 and scanning of arrays was performed using a GeneChip Scanner 3000 7G. The Affymetrix GeneChip Command Console (AGCC) software (v3.2) generated data cell files (DTC). For the process of RNA amplification, labeling, and hybridization, details are available from the Affymetrix website (https://www.affymetrix.com). Microarray scores for ER, PR, and HER2 were considered positive if ≥0.

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.