Jackson A Francina, Williams Andrew, Moffat Ivy, Phillips Suzanne L, Recio Leslie, Waters Michael D, Lambert Iain B, Yauk Carole L
Mechanistic Studies Division, Environmental Health Science and Research Bureau, Health Canada, Ottawa K1A 0K9, Canada; Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6, Canada.
Mechanistic Studies Division, Environmental Health Science and Research Bureau, Health Canada, Ottawa K1A 0K9, Canada.
J Pharmacol Toxicol Methods. 2013 Sep-Oct;68(2):260-268. doi: 10.1016/j.vascn.2013.02.008. Epub 2013 Mar 1.
Tissue samples are routinely formalin-fixed and paraffin-embedded (FFPE) for long term preservation. Gene expression analysis of archival FFPE tissues may advance knowledge of the molecular perturbations contributing to disease. However, formalin causes extensive degradation of RNA.
We compared RNA quality/yield from FFPE samples using six commercial FFPE RNA extraction kits. In addition we compared four DNA microarray protocols for the Agilent 8×60K platform using 16year old FFPE mouse liver samples treated with phenobarbital or vehicle.
Despite low quality RNA, archival phenobarbital samples exhibited strong induction of the positive control genes Cyp2b9 and Cyp2b10 by quantitative real-time PCR (qPCR). We tested one- and two-color microarray designs and evaluated the effects of increasing the amount of hybridized cDNA. Canonical gene responders to phenobarbital were measurably induced under each experimental condition. Increasing the amount of labeled cDNA did not improve the overall signal intensity. One-color experiments yielded larger fold changes than two-color and the number of differentially expressed genes varied between protocols. Gene expression changes were validated by qPCR and literature searches. Individual protocols exhibited high rates of false positives; however, pathway analysis revealed that nine of the top ten canonical pathways were consistent across experiments. Genes that were differentially expressed in more than one experiment were more likely to be validated. Thus, we recommend that experiments on FFPE samples be done in duplicate to reduce false positives.
In this analysis of archival FFPE samples we were able to identify pathways that are consistent with phenobarbital's mechanism of action. Therefore, we conclude that FFPE samples can be used for meaningful microarray gene expression analyses.
组织样本通常用福尔马林固定并石蜡包埋（FFPE）以长期保存。对存档的FFPE组织进行基因表达分析可能会增进对导致疾病的分子扰动的了解。然而，福尔马林会导致RNA大量降解。
我们使用六种商用FFPE RNA提取试剂盒比较了FFPE样本的RNA质量/产量。此外，我们使用经苯巴比妥或赋形剂处理的16年之久的FFPE小鼠肝脏样本，比较了安捷伦8×60K平台的四种DNA微阵列方案。
尽管RNA质量较低，但存档的苯巴比妥样本通过定量实时PCR（qPCR）显示出阳性对照基因Cyp2b9和Cyp2b10的强烈诱导。我们测试了单色和双色微阵列设计，并评估了增加杂交cDNA量的效果。在每种实验条件下，对苯巴比妥有典型反应的基因均可检测到诱导。增加标记cDNA的量并没有提高整体信号强度。单色实验产生的倍数变化比双色实验大，不同方案之间差异表达基因的数量也有所不同。基因表达变化通过qPCR和文献检索得到验证。个别方案显示出较高的假阳性率；然而，通路分析表明，十大典型通路中有九条在各实验中是一致的。在多个实验中差异表达的基因更有可能得到验证。因此，我们建议对FFPE样本进行的实验应重复进行以减少假阳性。
在对存档的FFPE样本的分析中，我们能够识别出与苯巴比妥作用机制一致的通路。因此，我们得出结论，FFPE样本可用于有意义的微阵列基因表达分析。
