Genespring gx version 13

CAFs were transfected with siRNAs as described above, and total RNA was extracted 72 h after transfection. Gene expression microarray analysis was then performed using a SurePrint G3 Human GE 8 × 60 K v2 microarray (Agilent Technologies, Santa Clara, CA, USA) as described previously [23 (link)]. Data were analyzed using GeneSpring GX version 13 (Agilent Technologies) and Gene set enrichment analysis (GSEA; Broad Institute, Boston, MA, USA). The Gene Expression Omnibus accession number for the microarray data is GSE234220.

OSCC cells were infected with lentivirus vectors as described above. Gene expression microarray analysis was carried out using SurePrint G3 Human GE microarray v3 (Agilent Technologies) as described previously^{41 (link)}. The microarray data were analyzed using GeneSpring GX version 13 (Agilent Technologies). The Gene Expression Omnibus accession number for the microarray data is GSE178613.

HCT116 cells were transfected with a ZNF582-AS1 expression vector or an empty vector as described above. Seventy-two hours after transfection, total RNA was extracted and labeled using a Quick Amp Labeling Kit One-Color (Agilent Technologies), after which, the synthesized cRNA was hybridized to the SurePrint G3 Human GE 8 × 60K V2 microarray (Agilent Technologies) according to the manufacturer’s instructions. The microarray data were analyzed using GeneSpring GX version 13 (Agilent Technologies).

Cells were transfected with siRNAs as described above, and total RNA was extracted 72 h after transfection. Gene expression microarray analysis was carried out according to the manufacturer’s instructions (Agilent Technologies, Santa Clara, CA, USA). Briefly, 100 ng of total RNA were amplified and labeled using a Low-input Quick Amp Labeling kit One-color (Agilent Technologies), after which the synthesized cRNA was hybridized to a SurePrint G3 Human GE microarray v2 (G4851; Agilent Technologies). The microarray data were analyzed using GeneSpring GX version 13 (Agilent Technologies). The Gene Expression Omnibus accession number for the microarray data is GSE108851.

GIST-T1 cells were transfected with miRNA mimics or a negative control as described above, and total RNA was extracted 48 h later. One-color microarray-based gene expression analysis was then carried out according to manufacturer’s instructions (Agilent Technologies). Briefly, 100 ng of total RNA were amplified and labeled using a Low-input Quick Amp Labelling kit One-color (Agilent Technologies), after which the synthesized cRNA was hybridized to a SurePrint G3 Human GE microarray v2 (G4851; Agilent Technologies). The microarray data were analyzed using GeneSpring GX version 13 (Agilent Technologies). The genes targeted by the miRNAs were predicted using the TargetScan system integrated into the GeneSpring GX software package. The Gene Expression Omnibus accession number for the microarray data is GSE68743.

The qualified RNA samples were labeled and hybridized on Agilent 4 × 44 K Custom design Gene Expression Microarray according to manufacturer protocol (Agilent, Santa Clara, CA). In brief, 100 ng of each RNA sample was amplified to cRNA and labeled with Cy-3 using Agilent Low Input Quick Amp Labeling Kit. The labeled samples were hybridized for 16 hours at 10 rotations per minute. After hybridization, the arrays were washed and scanned by Agilent scanner and then the image was analyzed by Agilent Feature Extraction v11.5. Data obtained from Feature Extraction were imported to GeneSpring GX version 13.1 (Agilent Technologies, Santa Clara, CA) for 75th percentile shift and baseline to median of all samples normalization.

The above RNA extracts were reverse transcribed to synthesize double stranded complementary DNA (cDNA). After purification of the double-stranded cDNA products, the sample was transcribed in vitro to generate Biotinylated complementary RNAs (cRNAs), followed by purification and fragmentation. The purified and fragmented cRNAs were then hybridised to the Affymetrix Soybean Gene Chip array (ThermoFisher Scientific, Lutterworth, UK). The scanned arrays produced CEL raw data files that were loaded onto Genespring GX version 13.1 (Agilent Genomics, Santa Clara, CA, USA) for further analysis. The extraction of genomic DNA (gDNA) from the two genotypes was performed using the DNA extraction Qiagen kit according to manufacturer’s instructions. Extracted DNA was labelled and hybridised to the Affymetrix Soybean TEST3 array and resulted in the generation of gDNA cell-intensity files (CEL files), after scanning. To identify probe pairs that efficiently hybridise to the gDNA, a series of user defined threshold values were evaluated for the signal intensity. The perfect match (PM) probes were selected for interpreting the GeneChip arrays challenged with RNA from the species of interest [35 (link)].