Human circular rna microarray

Profiling of circRNA expression in 100 human OFC postmortem brain (34 SCZ, 32 BD, and 34 Controls) was performed with the Arraystar Human Circular RNA Microarray (Arraystar Inc., Rockville, MD) per the manufacturer’s instructions with 13,617 probes designed to detect the unique circRNA splice junction based on numerous RNA-sequencing circRNA data [16 (link), 19 (link), 22 (link), 23 (link), 50 (link), 51 (link)]. Briefly 800 ng of total RNA previously quantified and quality verified (see above) were treated with an aggressive RnaseR treatment (3 h at 37 °C of ribonuclease R, 20 U/μL, Epicentre, Madison WI) to digest linear RNAs and enrich for circRNA expression. The enriched for circRNAs RNA was then amplified and transcribed into fluorescent cRNA via random primers according to the Arraystar Super RNA Labelling protocol (Arraystar Inc.). The labeled circRNAs were then hybridized onto the Arraystar Human Circular RNA arrays (8 × 15 K, Arraystar, Inc.) and incubated for 17 h at 65 °C in an Agilent hybridization oven (Agilent Technologies, Santa Clara, CA). Slides were then washed and scanned with the Agilent Scanner G2505C (Agilent Technologies). Differentially altered circRNAs as shown in Supplementary Tables 2–3. All circRNA profiling data have been deposited in the Mendeley online data repository: https://data.mendeley.com/datasets/9zdhc6pmx5/1.

The specimens used in the microarray were randomly selected from the 40 OC samples and 10 normal healthy control tissues described above. OC tissues were shock-frozen at once using liquid nitrogen. The specimens (three poly I:C-supplemented and three non-supplemented) were homogenized with TRIzol reagent (Invitrogen). NanoDrop ND-1000 was used to quantify total RNA in all the samples. The total RNA extracted from all the samples was amplified and transcribed to fluorescent circRNA using random primers and the Super RNA Labeling Kit (Arraystar Inc., Rockville, USA), according to the manufacturer’s instruction. Arraystar Human Circular RNA Microarray was used to hybridize the labeled circRNAs. Agilent G2505C Scanner (Agilent Technologies, California, USA) was used to scan arrays after washing the slides.