Surescan microarray scanner

The samples for microarrays were prepared according to the manufacturer’s instructions. These experiments were performed by Macrogen (Seoul, Korea). In briefly, the porcine (V2) gene expression 4x44K microarray (Agilent Technologies, Cat. no. G2519F-026440) and Illumina MouseRef-8 v2 Expression BeadChip (Illumina) was used. Labeling was carried out using a low RNA fluorescent linear amplification kit (Agilent Technologies, Cat. no. 5184-3523). The sample and control RNAs were labeled with Cy-3 and Cy-5, respectively for pig and biotin for mouse. Fragmentation was carried out by incubation at 60°C for 30 min in a fragmentation buffer, and the process was stopped by the addition of an equal volume of hybridization buffer. Hybridization was carried out at 60°C for 17 h in a hybridization oven. The hybridized array was scanned using a SureScan microarray scanner (Agilent Technologies, Cat. no. G4900DA) and Illumina Bead Array Reader Confocal Scanner. The TIFF image generated was loaded into the Feature Extraction software (Agilent Technologies, Cat. no. G4460AA) for feature data extraction, the data analyses were performed using the GeneSpring software (Agilent Technologies, Cat. no. G3778AA) and Illumina GenomeStudion v2011.1 (Gene Expression Module V1.9.0).

For studying the expression of miRs in the spinal tissue of rats, we sacrificed rats with the pentobarbital injection (n = 2 from each group). From each animal, the spinal cord tissue segment (10 mm) along with the injury epicenter was isolated and freezed in liquid nitrogen. The total RNA was separated from the spinal tissue with the help of TRIzol reagent (ThermoFisher, USA) following the supplied instructions, and the RNAs were purified using the RNeasy cleanup kit (Qiagen, Germany). The absorbance was measured using Shimadzu 1800 spectrophotometer (Shimadzu). The miRs were isolated using the microarray labeling kit (ThermoFisher, USA) and were then hybridized using the Exiqon miRCURY LNA array (Biocompare, USA). The slides were scanned using the SureScan microarray scanner (Agilent, USA). The miRs having an intensity of more than 50 were selected for further study, and the expression of miRs was normalized by median normalization. The miRs were evaluated by using volcano plots. The final clustering was utilized for determining the variations in the expression profiles of miRs.

An 8 × 60 K oligonucleotide array (Agilent Technologies) was used, and all procedures were performed according to the manufacturer's protocols.
A SurePrint G3 Human CGH Microarray Kit 8 × 60 K (Agilent Technologies) was used. DNA extraction was performed using a QIAamp DNA Blood Mini Kit (QIAGEN). Slides were scanned using the SureScan Microarray Scanner (Agilent Technology) and analyzed with Feature Extraction Software v11.5 (Agilent Technology) under the designed parameters of the human reference genome hg19. Data analysis was conducted using Agilent Cytogenomics software available on the company's website (https://www.genomics.agilent.com/en/CGH‐Microarray‐Data‐Analysis/CytoGenomics‐Software/?cid=AG‐PT‐111&tabId=AG‐PR‐1017, Agilent Cytogenomics v2.7.8.0).

Processing of purified RNA for microarray analysis and the microarray analysis were carried out at OakLabs (Hennigsdorf, Germany) according to their standard procedures. Briefly, RNA concentrations were between 17 and 66 ng/µL in volumes of 30 or 40 µL H₂O with integrity numbers (RIN, Bioanalyzer, Agilent Technologies, USA) between 6.3 and 7.9 (Supplementary Table S1). RNA was labeled using the Low-Input QuickAmp Labeling Kit (Agilent Technologies, USA) and cRNA was hybridized with ArrayXS Human (OakLabs, Germany) at 65 °C for 17 h using the Agilent Gene Expression Hybridization Kit (Agilent Technologies, USA), washed once with Agilent Gene Expression Wash Buffer 1 for one minute at room temperature, followed by a second wash with preheated (37 °C) Gene Expression Wash Buffer 2 for one minute.
Microarrays were scanned with a SureScan Microarray Scanner (Agilent Technologies, USA) and Agilent´s Feature Extraction software was used to detect features. Signals from control probes were removed and means of signals from replicate probes and of signals from all probes of a target were determined before normalization of the background subtracted signals. Data from all samples was quantile normalized using ranked mean quartiles [66 (link)]. Normalized data was statistically analyzed by paired analysis of variance (ANOVA) (clear cell foci vs. control samples from the same patient).

RNA samples from five PLC patients were analyzed using the Agilent 4 × 44 K Whole Human Genome Oligo microarrays (Agilent, cat.no G4112A). The labeling, hybridization, and washing were performed according to the manufacturer's instructions. Then, the slides were scanned using an Agilent SureScan Microarray Scanner (G2600D) and extracted with Agilent Feature Extraction Software v10.5.1.1.
The data were normalized using the quantile method from the “limma” R package. The raw and normalized PLC data were submitted to the GEO database with the accession number GSE92528.