Human clariom d

A total of 30 (18 males and 12 females, aged 35 ~ 81 years, without preoperative radiotherapy and chemotherapy) pairs of CRC specimens and adjacent para-tumoral normal tissues were collected from patients who underwent surgical resection at the Beijing Friendship Hospital, Capital Medical University from January, 2005 to December, 2012 (Supplementary information, Table S1). All cases were reviewed by a pathologist and diagnosed as CRC. Microarray analysis was performed on randomly selected 5 pairs of specimens using Affymetrix Clariom D Human with the support from Beijing Cnkingbio Biotechnology Corporation (China) (Supplementary information, Table S1). Briefly, the total RNA was extracted using TRIzol reagent (Life Technologies, USA) and purified with an RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. The RNA quantity and purity were determined by a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA) at the absorbance of 260 nm. The mRNA expression profiling was measured using Affymetrix Clariom D Human. RNA labeling, microarray hybridization and scanning were performed according to the manufacturer’s instructions. The arrays were scanned using the Gene-Chip® scanner 3000 (Affymetrix, USA). GeneChip operating software 1.4 was then used to analyze the array data. The differentially expressed genes were defined as > twofold change.

Total RNA extraction from peripheral blood mononuclear cells (PBMCs) was performed using a miRNeasy mini kit following the manufacturer’s protocol (Qiagen GmbH, Hilden, Germany). Furthermore, cRNA preparation, sample hybridization, and scanning were performed following the protocols provided by Affymetrix (Affymetrix, Santa Clara, CA, USA) and Cogentech Affymetrix microarray unit (Campus IFOM IEO, Milan, Italy). All samples were hybridized on a Human Clariom D (Thermo Fisher Scientific) gene chip and were analyzed using the Transcriptome Analysis Console 4.0 software (Applied Biosystem, Foster City, CA, USA by Thermo Fisher Scientific, Waltham, MA, USA). Human Clariom D arrays enable investigation of more than 540,000 transcripts sourced from the largest public databases starting from as little as 100 pg of total RNA. Relative gene expression levels of each transcript were validated by applying a one-way analysis of variance (p ≤ 0.01) and multiple testing corrections. Coding genes and lncRNAs that displayed an expression level at least 1.5-fold different in the test sample versus control sample (p ≤ 0.01) were carried forward in subsequent analyses.