Gene signature predictive of response to chemotherapy in mCRC [RT-PCR]. Homo sapiens

The objective is to generate a robust and validated predictor profile for chemotherapy response in patients with mCRC using microarray gene expression profiles of primary colorectal cancer tissue. Overall design: To define a gene signature of response to chemotherapy in metastatic colorectal cancer, samples were obtained from 40 patients from Marques de Valdecilla Hospital who underwent primary surgery. Gene expression was detected and quantified using the Human Whole Genome U133 Plus 2.0 array (Affymetrix), containing 54675 human gene probes. The validation set consisted of 119 samples from Hospital Virgen del Rocio, Seville, Spain; Hospital Virgen de la Victoria, Malaga, Spain; Hospital de la Merced, Osuna, Spain and Hospital Marqués de Valdecilla, Santander, Spain, and included 86 tumor samples (40 coming from the training set and 46 from newly treated CRC patients) and 33 normal tissue samples of CRC patients used as controls. Custom-designed TaqMan® Low Density Arrays (TLDA) 7900 HT Micro Fluidic Cards including the 161 genes selected for validation were run and analyzed by the ABI PRISM® 7900HT Sequence Detection System (SDS 2.2, Applied Biosystems) according to manufacturer's protocol (Applied Biosystems). Expression of target miRNAs was normalized in relation to the expression of GAPDH. Cycle threshold (Ct) values were calculated using the SDS software v.4.2 using automatic baseline settings and a threshold of 0.2. Relative quantification of gene expression was calculated by the 2−ΔCt method (Applied Biosystems user bulletin no. 2 (P/N 4303859)). This submission represents the RT-PCR component of the study only

本研究旨在利用结直肠癌原发组织的微阵列基因表达谱，构建针对转移性结直肠癌（metastatic colorectal cancer, mCRC）患者化疗响应性的稳健且经过验证的预测特征谱。
整体实验设计：为明确转移性结直肠癌化疗响应的基因特征，研究纳入了来自西班牙桑坦德马尔克斯德巴尔塞迪亚医院（Marqués de Valdecilla Hospital）的40名接受原发肿瘤切除术的患者样本。采用人类全基因组U133 Plus 2.0芯片（Affymetrix，包含54675个人类基因探针）对基因表达进行检测与定量。
验证集样本取自西班牙塞维利亚维尔京德尔罗西奥医院（Hospital Virgen del Rocio）、马拉加维尔京德拉维多利亚医院（Hospital Virgen de la Victoria）、奥苏纳拉梅塞德医院（Hospital de la Merced）以及桑坦德马尔克斯德巴尔塞迪亚医院，共计119例样本，其中包含86例肿瘤样本（40例来自训练集，46例为新入组的结直肠癌患者样本）与33例结直肠癌患者正常组织对照样本。
针对筛选出的161个用于验证的基因，研究采用定制设计的TaqMan®低密度阵列（TLDA）7900 HT微流体卡，并通过ABI PRISM® 7900HT序列检测系统（SDS 2.2，应用生物系统公司（Applied Biosystems））按照厂商操作规程进行上机检测与数据分析。
靶标miRNA的表达以甘油醛-3-磷酸脱氢酶（GAPDH）的表达作为内参进行归一化处理。
采用SDS软件v.4.2，以自动基线设置及阈值0.2计算循环阈值（Ct值）。
基因表达的相对定量采用2^(-ΔCt)法计算（参考应用生物系统公司用户手册第2号（产品编号P/N 4303859））。
本提交仅涵盖本研究的逆转录聚合酶链反应（Reverse Transcription-Polymerase Chain Reaction, RT-PCR）实验部分。