Agilent hybridization oven

For microarrays, the synthesis of target cRNA probes and hybridization were performed using Agilent’s Low RNA Input Linear Amplification kit (Agilent Technologies, Palo Alto, CA, USA) according to the manufacturer’s instructions. The fragmented cRNA was resuspended with 2X hybridization buffer and directly pipetted onto the assembled Agilent’s Bovine Oligo Microarray (44K). The arrays were hybridized at 65°C for 17 h using the Agilent Hybridization oven and the hybridized microarrays were washed as described in the manufacturer’s washing protocol (Agilent Technologies).
The hybridized images were scanned using Agilent’s DNA microarray scanner and quantified with Feature Extraction Software (Agilent Technologies). All data normalization and selection of fold-changed genes were performed using GeneSpringGX 7.3 (Agilent Technologies). The averages of normalized ratios were calculated by dividing the average of the normalized signal channel intensity by the average of the normalized control channel intensity. Hierarchical clustering was performed with TIGR MeV Ver.4.9 software (Institute of Genomic Research, Rockville, MD, USA) [26 (link)]. Microarray data are available from Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) with the accession number GSE92672.

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.

For each sample,
a predetermined amount of synthetic DNA was added to 1 μg of
extracted DNA as a spike-in control. Subsequently, the mixed DNA was
labeled with Cy-3 (or Cy-5) fluorescent dye (GE Healthcare, Vacaville,
CA, USA) using random primers and Klenow fragment of DNA polymerase
I. Labeled DNA was then purified using a QIAquick Purification kit
(Qiagen, Valencia, CA, USA), and the NanoDrop 8000 UV–vis Spectrophotometer
(Thermo Scientific; Waltham, MA) was used to measure the yield and
degree of labeling. Each sample was supplemented with a total of 42 μL
of buffer containing 1× HI-RPM hybridization buffer, 1×
aCGH blocking agent, 0.05 μg/μL of Cot-1 DNA, and 10%
formamide. The mixture was then vortexed thoroughly, spun down, and
incubated at 95 °C for 3 min, followed by incubation at 37 °C
for 30 min. The samples were subsequently hybridized with CyanoStrainChip
at 67 °C for 24 h with a rotation at 20 rpm in an Agilent hybridization
oven (Agilent Technologies, Inc., Santa Clara, CA, USA). For posthybridization
washing, an Agilent Wash Buffer Kit (Agilent, Santa Clara, CA) was
used for removing nonhybridized or partially hybridized labeled sample
DNA from the array’s surface to minimize signal noise.

Samples were labeled and array hybridization was performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology). The total RNA from each sample was linearly amplified and labeled with Cy3–UTP. The labeled cRNAs were purified with the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. The concentration and specific activity of the labeled cRNAs (pmol Cy3/µg cRNA) were measured with a NanoDrop ND-1000 spectrophotometer. Each labeled cRNA (1 µg) was fragmented by the addition of 11 µl of 10× Blocking Agent and 2.2 µl of 25× Fragmentation Buffer and was then heated at 60°C for 30 min. Next, 55 µl of 2× GE Hybridization Buffer was added to dilute the labeled cRNA. The hybridization solution (100 µl) was dispensed onto the gasket slide, which was assembled onto the gene expression microarray slide. The slides were incubated for 17 h at 65°C in an Agilent Hybridization Oven. The hybridized arrays were washed, fixed, and scanned with the Agilent DNA Microarray Scanner.

Three gastric cancer tissues and their matched adjacent nontumorous tissues 5 cm away from the edge of tumor were selected to analyze circRNA expression profile using Arraystar Human circRNA Array (Arraystar, Rockville, MD). Total RNA from six samples were amplified and transcribed into fluorescent cRNA utilizing random primer according to Arraystar's Super RNA Labeling protocol (Arraystar). The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array (6 × 7K, Arraystar), and incubated for 17 h at 65°C in an Agilent Hybridization Oven (Agilent Technologies, Santa Clara, CA). After having washed the slides, the arrays were scanned by the Axon GenePix 4000B microarray scanner (Molecular Devices, Sunnyvale, CA).
Scanned images were then imported into GenePix Pro 6.0 software (Axon) for grid alignment and data extraction. Quantile normalization and subsequent data processing were performed using the R software package. Differentially expressed circRNAs with statistical significance between two groups were identified through Fold Change filtering or Volcano Plot filtering. Hierarchical clustering was performed to show the distinguishable circRNA expression pattern among samples.

Isolated total RNA was treated with Rnase R (Epicentre, Inc.) to remove linear RNA, and then was amplified and transcribed into fluorescent cRNA using random primer according to Arraystar Super RNA Labeling protocol. Next, the labeled cRNA was purified by RNeasy Mini Kit (Qiagen, Germany). Specific activity (pmol Dye/μg cRNA) and concentration (μg/μl) of cRNA were surveyed to assess the labeling efficiency by Nano Drop ND-1000. After cRNA preparation, 1 μg labeled cRNA, mixed with 1 μl 25× Fragmentation Buffer and 5 μl 10× Blocking Agent, was heated at 60 °C for half an hour. After deliquating the labeled cRNA with 25 μl 2× Hybridization buffer, 50 μl hybridization solution was added into the gasket slide and assembled to circRNA expression microarray. The slide was incubated at 65 °C for seventeen hours in Agilent Hybridization Oven. After washing, slides were fixed and scanned to generate images using Agilent Scanner G2505C. And acquired array images were analyzed by Agilent Feature Extraction software. The microarray hybridization and the collection of data were performed by KangChen Bio-tech, Shanghai, China.