Pathogenetics Unit, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health Bethesda, Maryland, USA.
Am J Cancer Res. 2013 Aug 14;3(4):402-10. eCollection 2013.
In a recent study, a unique gene expression signature was observed when comparing esophageal squamous cell carcinoma (ESCC) epithelial cells to normal esophageal epithelial cells using laser capture microdissection (LCM) and cDNA microarray technology. To validate the expression of several intriguing genes from that study (KRT17, cornulin, CD44, and EpCAM), we employed two new technologies, expression microdissection (xMD) for high-throughput microdissection facilitating protein analysis and RNAscope for the evaluation of low abundant transcripts in situ. For protein measurements, xMD technology was utilized to specifically procure sufficient tumor and normal epithelium from frozen human tissue for immunoblot analysis of KRT17 (CK17) and cornulin. A novel in situ hybridization method (RNAscope) was used to determine the transcript level of two relatively low expressed genes, CD44 and EpCAM in both individual formalin-fixed paraffin-embedded (FFPE) tissue sections and in an ESCC tissue microarray (TMA). The results successfully confirmed the initial expression pattern observed for all four genes, potentially implicating them in the pathogenesis of ESCC. Additionally, the study provides important methodological information on the overall process of candidate gene validation.
在最近的一项研究中，通过激光捕获显微切割（LCM）和 cDNA 微阵列技术，将食管鳞状细胞癌（ESCC）上皮细胞与正常食管上皮细胞进行比较，观察到一个独特的基因表达特征。为了验证该研究中几个有趣基因（KRT17、cornulin、CD44 和 EpCAM）的表达，我们采用了两种新技术，即表达微切割（xMD）用于高通量微切割以促进蛋白质分析，以及 RNAscope 用于原位评估低丰度转录本。对于蛋白质测量，xMD 技术用于从冷冻人组织中特异性获取足够的肿瘤和正常上皮细胞，以进行 KRT17（CK17）和 cornulin 的免疫印迹分析。一种新的原位杂交方法（RNAscope）用于确定两个相对低表达基因 CD44 和 EpCAM 在单个福尔马林固定石蜡包埋（FFPE）组织切片和 ESCC 组织微阵列（TMA）中的转录本水平。结果成功地证实了所有四个基因最初观察到的表达模式，这可能暗示它们参与了 ESCC 的发病机制。此外，该研究还提供了有关候选基因验证总体过程的重要方法学信息。
