Gene expression profiling study to identify key markers associated with disease recurrence in colorectal cancers.

The objective of this study is to identify a gene set to predict recurrence of colorectal cancer (CRC) patients. We generated RNA-seq data of 110 primary CRC samples and identified significant genes associated with recurrence of CRC. Through diverse statistical methods including generalized linear model likelihood ratio test, significant 10 genes were identified. In the validation cohorts, a risk classifier consisting of the 10 genes was an independent risk factor in colorectal cancers. Total RNA was isolated using an RNeasy Mini Kit (Qiagen, CA, USA) according to the manufacturer’s protocol. The quality and integrity of the RNA were confirmed by agarose gel electrophoresis and ethidium bromide staining, followed by visual examination under ultraviolet light. The sequencing library was prepared using a TruSeq RNA Sample Preparation kit v2 (Illumina, CA, USA) according to the manufacturer’s protocol. In brief, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, fragmented and converted into cDNA. After the ligation of adapters, the fragments were amplified by PCR. Sequencing was performed on paired end reads (2×100 bp) using the HiSeq-2000 platform (Illumina).