Dietrich Dimo, Lesche Ralf, Tetzner Reimo, Krispin Manuel, Dietrich Jörn, Haedicke Wolfgang, Schuster Matthias, Kristiansen Glen
Epigenomics AG, Kleine Präsidentenstr. 1, 10178 Berlin, Germany.
J Histochem Cytochem. 2009 May;57(5):477-89. doi: 10.1369/jhc.2009.953026. Epub 2009 Jan 19.
A procedure for simultaneous quantification of DNA methylation of several genes in minute amounts of sample material was developed and applied to microdissected formalin-fixed and paraffin-embedded breast tissues. The procedure is comprised of an optimized bisulfite treatment protocol suitable for samples containing only few cells, a multiplex preamplification and subsequent locus specific reamplification, and a novel quantitative bisulfite sequencing method based on the incorporation of a normalization domain into the PCR product. A real-time PCR assay amplifying repetitive elements was established to quantify low amounts of bisulfite-treated DNA. Ten prognostic and diagnostic epigenetic breast cancer biomarkers (PITX2, RASSF1A, PLAU, LHX3, PITX3, LIMK1, SLITRK1, SLIT2, HS3ST2, and TFF1) were analyzed in tissue samples obtained from two patients with invasive ductal carcinoma of the breast. The microdissected samples were obtained from several areas within the tumor tissue, including intraductal and invasive carcinoma, adenosis, and normal ductal epithelia of adjacent normal tissue, as well as stroma, tumor infiltrating lymphocytes, and adipose tissue. Overall, reliable quantification was possible for all genes. For most genes, increased DNA methylation in invasive and intraductal carcinoma cells compared with other tissue components was observed. For TFF1, decreased methylation levels were observed in tumor cells.
我们开发了一种可同时对微量样本材料中的多个基因进行DNA甲基化定量的方法，并将其应用于显微切割的福尔马林固定石蜡包埋乳腺组织。该方法包括适用于仅含少量细胞样本的优化亚硫酸氢盐处理方案、多重预扩增及随后的位点特异性再扩增，以及一种基于在PCR产物中引入标准化结构域的新型定量亚硫酸氢盐测序方法。建立了一种扩增重复元件的实时PCR检测方法，以定量少量经亚硫酸氢盐处理的DNA。对两名乳腺浸润性导管癌患者的组织样本分析了10种预后和诊断性乳腺癌表观遗传生物标志物（PITX2、RASSF1A、PLAU、LHX3、PITX3、LIMK1、SLITRK1、SLIT2、HS3ST2和TFF1）。显微切割样本取自肿瘤组织内的多个区域，包括导管内癌和浸润癌、腺病、相邻正常组织的正常导管上皮，以及基质、肿瘤浸润淋巴细胞和脂肪组织。总体而言，所有基因均可进行可靠的定量分析。对于大多数基因，与其他组织成分相比，浸润性和导管内癌细胞中的DNA甲基化增加。对于TFF1，在肿瘤细胞中观察到甲基化水平降低。
