Liu B H, Goh C H K, Ooi L L P J, Hui K M
Bek Chai Heah Laboratory of Cancer Genomics, Division of Cellular and Molecular Research, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore, Singapore.
Oncogene. 2008 Jul 3;27(29):4128-36. doi: 10.1038/onc.2008.50. Epub 2008 Mar 10.
Most human cancers are characterized by genetic aberrations accompanied by altered expression and function of numerous genes. Applying genome-wide, microarray gene expression analysis to identify deregulated genes in different tumour types can provide potential gene candidates as diagnostic and prognostic tools and promising targets for drug development. However, the detection of deregulated genes with low levels of expression remains a major challenge. In this study, we have designed a strategy, termed modified suppression subtractive hybridization (mSSH), to identify genes encoding rare transcripts. The strategy entails incorporating the T(7)-promoter sequence at the 5' end of the noncoding cDNA strand during first strand cDNA synthesis to generate unidirectional antisense RNA from the resultant cDNA following conventional SSH. These transcripts are subsequently analysed by Affymetrix oligonucleotide gene arrays. Here, we have used five hepatocellular carcinoma (HCC), five breast carcinoma and four nasopharyngeal carcinoma (NPC) biopsies separately as testers and their corresponding normal biopsies as drivers to enrich for low abundance tumour type-specific transcripts. The total detectable number of probe sets following mSSH was reduced almost 10-fold in comparison to those detected for the same resected tumour tissues without undergoing subtraction, thus yielding a subtraction efficacy of over 90%. Using this experimental approach, we have identified 48 HCC-specific, 45 breast carcinoma-specific, and 83 NPC-specific genes. In addition, 115 genes were upregulated in all the three cancer types. When compared to gene-profiling data obtained without mSSH, the majority of these identified transcripts were of low abundance in the original cancer tissues. mSSH can therefore serve as a comprehensive molecular strategy for pursuing functional genomic studies of human cancers.
大多数人类癌症的特征是基因畸变，同时伴随着众多基因的表达和功能改变。应用全基因组微阵列基因表达分析来识别不同肿瘤类型中失调的基因，可为诊断和预后提供潜在的基因候选物，并为药物开发提供有前景的靶点。然而，检测低表达水平的失调基因仍然是一项重大挑战。在本研究中，我们设计了一种称为改良抑制消减杂交（mSSH）的策略，以识别编码稀有转录本的基因。该策略包括在第一链cDNA合成过程中，将T(7)启动子序列掺入非编码cDNA链的5'端，以便在常规SSH后从所得cDNA产生单向反义RNA。随后通过Affymetrix寡核苷酸基因阵列分析这些转录本。在这里，我们分别使用五例肝细胞癌（HCC）、五例乳腺癌和四例鼻咽癌（NPC）活检组织作为测试样本，以相应的正常活检组织作为驱动样本，来富集低丰度的肿瘤类型特异性转录本。与未进行消减的相同切除肿瘤组织相比，mSSH后可检测到的探针集总数减少了近10倍，从而产生了超过90%的消减效率。通过这种实验方法，我们鉴定出了48个HCC特异性基因、45个乳腺癌特异性基因和83个NPC特异性基因。此外，有115个基因在所有这三种癌症类型中均上调。与未使用mSSH获得的基因谱数据相比，这些鉴定出的转录本在原始癌组织中大多为低丰度。因此，mSSH可作为一种全面的分子策略，用于开展人类癌症的功能基因组研究。
