Sureprint g3 human gene expression 8x60k v2 microarray

DNA and RNA isolation and the GEP and WES analyses were performed as described previously (20 (link)). RNA samples with an RNA integrity number ≥6.0 were used for microarray analysis. Labeled samples were hybridized to the SurePrint G3 Human Gene Expression 8x60 K v2 Microarray (Agilent Technologies). Microarray analysis was performed in accordance with the MIAME guidelines. For DNA data analysis, somatic mutations were identified by comparing data from tumor and corresponding blood samples. Mutations in 138 known driver genes were defined as those identified as pathogenic in the ClinVar database. Vogelstein et al (21 (link)) demonstrated that 138 genes, when altered by intragenic mutations, can promote or drive tumorigenesis. A most of tumors including colorectal cancers contain two to eight of these ʻdriver gene’ mutations and the remaining mutations are passengers that do not contribute to tumorigenesis directly. Thus, these 138 driver mutations are accepted as relevant genes to the tumorigenesis (21 (link)). Single nucleotide variants (SNVs) of the total exonic mutations for each sequenced tumor included nonsynonymous, synonymous, and indel/frameshift mutations.

Microarray expression profiling was conducted in liver tissue from healthy
controls (n = 6) and in patients with liver hydatid disease (n = 6). Briefly,
total RNA (100 ng) was labeled with the mRNA Complete Labeling and Hyb Kit
(Agilent Technologies) and hybridized on the SurePrint G3 Human Gene Expression
8x60K v2 microarray (Agilent Technologies). The microarray contains 50,599
probes for 32,776 human mRNAs originating from authoritative databases,
including RefSeq, Ensemble, GenBank, and the Broad Institute. After
hybridization and washing, processed slides were scanned with the Agilent G2505C
microarray scanner (Agilent Technologies).

Eight samples (tumour n = 4, nontumour n = 4) were considered to have acceptable RNA quality for microarray analysis (RIN > 6·5, 260/280 and 260/230 > 1·8). From each sample, 25 ng of total RNA was used for the generation of amplified, fluorescent complementary RNA (cRNA), labelled with cyanine dye (Cy3) with the one‐color Low Input Quick Amp Labeling Kit (Agilent Technologies). The cRNA was purified using the RNeasy Mini Kit (Qiagen), and hybridized to SurePrint G3 Human Gene Expression 8x60K v2 Microarrays (Agilent Technologies) for 17 h at 65 °C before the fluorescent pattern was read with a G2565CA Microarray Scanner (Agilent Technologies). Analysis of the raw microarray images was performed using Agilent Feature Extraction software v10·7·3·1 (Agilent Technologies).