Rna mini kit

The structures were homogenized with Bioprep-24 Homogenizer (Aosheng, Hangzhou, China). The RNA Mini Kit (A&A Biotechnology, Gdańsk, Poland) was used to isolate RNA according to the instructions. The total RNA was reverse transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). RT-qPCR was performed in duplicate on a 96-well plate using QuantStudio 3 (Applied Biosystems). Gene expression was assessed with the following TaqMan Gene Expression Assays (Thermo Fisher Scientific, Waltham, MA, USA): Wnt5a (assay ID: Rn0140000_m1), Wnt7b (assay ID: Rn01504313_m1) and Ctnnb1 (assay ID: Rn00670330_m1). The following conditions were used: an initial step of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and then 60 °C for 60 s. The 18S rRNA (assay ID: 99999901) was used as housekeeping controls, and the relative expression of target genes was calculated by comparative Ct method (2^−ΔΔCt), and values are expressed as the fold change from the control (yoked saline group).

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.

Total RNA was isolated from human thyroid specimens and thyroid cancer cell lines using an RNA Mini Kit (A&A Biotechnology, Poland) according to the recommended protocol, and the RNA integrity was verified by agarose gel electrophoresis. Total RNA (1 µg) was used for cDNA synthesis with a High Capacity cDNA Reverse Transcription Kit (Life Technologies, Applied Biosystems, USA). Expression of the human PDPN and 18S rRNA genes was quantified by RT-qPCR using the cDNAs as template in reactions containing the double-stranded DNA-specific dye SYBR Green I and Maxima Fluorescein RT-qPCR Master Mix (Thermo Scientific, Canada), and specific oligonucleotide primers (listed below), as described previously [24] (link).
PDPN (NM_006474)
Forward: 5′-CGAAGATGATGTGGTGACTC-3′Reverse: 5′-CGATGCGAATGCCTGTTAC-3′18S rRNA (NM_02255)
Forward: 5′-CCAGTAAGTGCGGGGTCATAAG-3′Reverse: 5′-CCATCCAATCGGTAGTAGCG-3′.
Amplification, data acquisition and data analysis were performed using the iQ5 Real-Time PCR Detection System and software (Bio-Rad, USA).