Genechip mirna 4.0 array process

The Affymetrix GeneChip miRNA 4.0 array process was carried out according to the manufacturer’s protocol (Affymetrix, Santa Clara, CA, USA). Approximately 1,000 ng of RNA was labeled with the FlashTag Biotin RNA Labeling Kit (Genisphere, Hatfield, PA, USA). The labeled RNA was then quantified, fractionated, and hybridized to the miRNA microarray chip according to the standard procedures recommended by the manufacturer. The labeled RNA was heated to 99°C for 5 minutes and then to 45°C for another 5 minutes. RNA-array hybridization was performed in an Affymetrix Fluidics Station 450 with agitation at 60 rotations per minute for 16 hours at 48°C. The chips were washed and stained using a GeneChip Fluidics Station 450 (Affymetrix) and then scanned with an Affymetrix GCS 3000 scanner. Signals were quantified using the Affymetrix GeneChip Command Console (AGCC) software.

The Affymetrix Genechip miRNA 4.0 array process was executed according to the manufacturer's protocol. 1000 ng RNA samples were labeled with the FlashTag™ Biotin RNA Labeling Kit (Genisphere, Hatfield, PA, USA). The labeled RNA was quantified, fractionated and hybridized to the miRNA microarray according to the standard procedures provided by the manufacture. The labeled RNA was heated to 99°C for 5 minutes and then to 45 °C for 5 min.
RNA-array hybridization was performed with agitation at 60 rotations per minute for 16 h at 48 °C on an Affymetrix GeneChip Hybridization oven 645. The chips were washed and stained using a Genechip Fluidics Station 450 (Affymetrix, CA, United States). The chips were then scanned with an Affymetrix GCS 3000 canner (Affymetrix).
MiRNA-Gene ontology (GO) analysis was performed by top 5 list of miRNAs that satisfy |fold change| ≥ 2 and p value < 0.05 between primary CRC group and paired PTM group. The miRNA-GO analysis was conducted through DIANA-mirPath v 3.0 software.
The genes targeted by miR-193a used heatmap to compare the expression levels in each CRC-PTM cell line set (SNU-2335A, SNU-2335D, SNU-2404A, SNU-2404B, SNU-2414A and SNU-2414B) and the heatmap were analyzed using “pheatmap” libarary in R program (v 4.0.0).