Biomolecular Screening Branch, Division of the National Toxicology Program, National Institute of Environmental Health Sciences , Research Triangle Park, NC 27709, United States.
Chem Res Toxicol. 2012 May 21;25(5):1132-44. doi: 10.1021/tx3000945. Epub 2012 May 4.
Archival tissues from laboratory studies represent a unique opportunity to explore the relationship between genomic changes and agent-induced disease. In this study, we evaluated the applicability of qPCR for detecting genomic changes in formalin-fixed, paraffin-embedded (FFPE) tissues by determining if a subset of 14 genes from a 90-gene signature derived from microarray data and associated with eventual tumor development could be detected in archival liver, kidney, and lung of rats exposed to aflatoxin B1 (AFB1) for 90 days in feed at 1 ppm. These tissues originated from the same rats used in the microarray study. The 14 genes evaluated were Adam8, Cdh13, Ddit4l, Mybl2, Akr7a3, Akr7a2, Fhit, Wwox, Abcb1b, Abcc3, Cxcl1, Gsta5, Grin2c, and the C8orf46 homologue. The qPCR FFPE liver results were compared to the original liver microarray data and to qPCR results using RNA from fresh frozen liver. Archival liver paraffin blocks yielded 30 to 50 μg of degraded RNA that ranged in size from 0.1 to 4 kB. qPCR results from FFPE and fresh frozen liver samples were positively correlated (p ≤ 0.05) by regression analysis and showed good agreement in direction and proportion of change with microarray data for 11 of 14 genes. All 14 transcripts could be amplified from FFPE kidney RNA except the glutamate receptor gene Grin2c; however, only Abcb1b was significantly upregulated from control. Abundant constitutive transcripts, S18 and β-actin, could be amplified from lung FFPE samples, but the narrow RNA size range (25-500 bp length) prevented consistent detection of target transcripts. Overall, a discrete gene signature derived from prior transcript profiling and representing cell cycle progression, DNA damage response, and xenosensor and detoxication pathways was successfully applied to archival liver and kidney by qPCR and indicated that gene expression changes in response to subchronic AFB1 exposure occurred predominantly in the liver, the primary target for AFB1-induced tumors. We conclude that an evaluation of gene signatures in archival tissues can be an important toxicological tool for evaluating critical molecular events associated with chemical exposures.
实验室研究的存档组织代表了一个独特的机会，可以探索基因组变化与诱导疾病的物质之间的关系。在这项研究中，我们通过确定从微阵列数据中得出的与最终肿瘤发展相关的 90 个基因特征中的一小部分基因（14 个基因）是否可以在喂食 1ppm 黄曲霉毒素 B1（AFB1）90 天的大鼠的存档肝、肾和肺中检测到，评估了 qPCR 检测福尔马林固定、石蜡包埋（FFPE）组织中基因组变化的适用性。这些组织源自用于微阵列研究的同一批大鼠。评估的 14 个基因是 Adam8、Cdh13、Ddit4l、Mybl2、Akr7a3、Akr7a2、Fhit、Wwox、Abcb1b、Abcc3、Cxcl1、Gsta5、Grin2c 和 C8orf46 同源物。qPCR FFPE 肝脏结果与原始肝脏微阵列数据以及使用新鲜冷冻肝脏 RNA 的 qPCR 结果进行了比较。存档肝脏石蜡块产生了 30 到 50 μg 的降解 RNA，大小范围从 0.1 到 4 kB。通过回归分析，FFPE 和新鲜冷冻肝脏样本的 qPCR 结果呈正相关（p≤0.05），并且对于 14 个基因中的 11 个，qPCR 结果与微阵列数据在变化的方向和比例上具有良好的一致性。除谷氨酸受体基因 Grin2c 外，所有 14 个转录本均可从 FFPE 肾 RNA 中扩增，但仅 Abcb1b 从对照中显著上调。丰度组成型转录物 S18 和 β-肌动蛋白可从肺 FFPE 样本中扩增，但目标转录物的 RNA 大小范围狭窄（25-500 bp 长度）阻止了一致的检测。总体而言，从先前的转录谱中获得的离散基因特征，代表细胞周期进展、DNA 损伤反应以及 Xenensor 和解毒途径，通过 qPCR 成功应用于存档肝和肾，并表明对亚慢性 AFB1 暴露的基因表达变化主要发生在肝脏，这是 AFB1 诱导肿瘤的主要靶标。我们得出结论，对存档组织中的基因特征进行评估可以成为评估与化学暴露相关的关键分子事件的重要毒理学工具。
