Gene expression profiling study by RNA-seq to identify gene signature resposed to chemo-radiotherapy in colorectal cancers.. Homo sapiens

The objective of this study is to assess gene expression changes and identify gene sets associated with chemo-radiotherapy (CRT) in colorectal cancer (CRC) patients. We performed RNA-seq based transcriptome profiling using primary tumor samples obtained from 22 CRC patients. By a number of statistical tests, a total of 575 genes were statistically significant, indicating a gene set profoundly responded to chemo-radiotherapy in CRC. To select the genes showing strong gene expression intensities and critically validate the differences between treatment-responded and non-responded patients, we applied more rigorous cut-off for gene selection, revealing a total of 8 final gene candidates. These genes were applied to the downstream experimental assays. Overall design: RNA-seq data of 22 CRC samples were generated. Total RNA was isolated by 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. Sequencing library was prepared using TruSeq RNA Sample Preparation kit v2 (Illumina, CA, USA) according to the manufacturer’s protocols. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, fragmented, and converted into cDNAs. Then, adapters were ligated and the fragments were amplified on a PCR. Sequencing was performed in paired end reads (2x100 bp) using Hiseq-2000 (Illumina).

本研究旨在评估结直肠癌（colorectal cancer, CRC）患者接受放化疗（chemo-radiotherapy, CRT）后的基因表达变化，并筛选与之相关的基因集。我们收集了22名结直肠癌患者的原发性肿瘤样本，开展了基于RNA测序（RNA-seq）的转录组谱分析（transcriptome profiling）。经多项统计学检验，共筛选出575个具有统计学显著性的基因，提示存在一组对结直肠癌放化疗产生显著响应的基因集。为筛选出高表达强度的基因，并严格验证治疗响应者与非响应者之间的表达差异，我们采用了更为严格的基因筛选截断阈值（cut-off），最终得到8个候选基因。上述基因被应用于后续的实验检测。总体实验设计：本研究生成了22份结直肠癌样本的RNA测序数据。总RNA依照制造商说明书，使用RNeasy Mini试剂盒（RNeasy Mini Kit, Qiagen，美国加利福尼亚州）进行提取。RNA的质量与完整性通过琼脂糖凝胶电泳（agarose gel electrophoresis）结合溴化乙锭染色（ethidium bromide staining）进行验证，并在紫外灯下进行目视检查。测序文库依照制造商方案，采用TruSeq RNA Sample Preparation kit v2（Illumina，美国加利福尼亚州）制备。简要流程如下：利用寡聚dT磁珠（poly-T oligo-attached magnetic beads）从总RNA中纯化mRNA，将其片段化并反转录为互补DNA（cDNA）；随后连接测序接头（adapters），通过聚合酶链式反应（PCR）扩增得到测序文库片段。测序采用Illumina Hiseq-2000平台完成双端测序读段（paired end reads, 2×100 bp）。