Genomic Health Inc, Redwood City, California, United States of America.
PLoS One. 2012;7(7):e40092. doi: 10.1371/journal.pone.0040092. Epub 2012 Jul 13.
RNA biomarkers discovered by RT-PCR-based gene expression profiling of archival formalin-fixed paraffin-embedded (FFPE) tissue form the basis for widely used clinical diagnostic tests; however, RT-PCR is practically constrained in the number of transcripts that can be interrogated. We have developed and optimized RNA-Seq library chemistry as well as bioinformatics and biostatistical methods for whole transcriptome profiling from FFPE tissue. The chemistry accommodates low RNA inputs and sample multiplexing. These methods both enable rediscovery of RNA biomarkers for disease recurrence risk that were previously identified by RT-PCR analysis of a cohort of 136 patients, and also identify a high percentage of recurrence risk markers that were previously discovered using DNA microarrays in a separate cohort of patients, evidence that this RNA-Seq technology has sufficient precision and sensitivity for biomarker discovery. More than two thousand RNAs are strongly associated with breast cancer recurrence risk in the 136 patient cohort (FDR <10%). Many of these are intronic RNAs for which corresponding exons are not also associated with disease recurrence. A number of the RNAs associated with recurrence risk belong to novel RNA networks. It will be important to test the validity of these novel associations in whole transcriptome RNA-Seq screens of other breast cancer cohorts.
通过对存档福尔马林固定石蜡包埋（FFPE）组织进行基于 RT-PCR 的基因表达谱分析发现的 RNA 生物标志物，构成了广泛使用的临床诊断测试的基础；然而，RT-PCR 在可检测的转录本数量上受到实际限制。我们已经开发和优化了 RNA-Seq 文库化学以及用于 FFPE 组织的全转录组分析的生物信息学和生物统计学方法。该化学方法可适应低 RNA 输入和样品多路复用。这些方法都能够重新发现通过对 136 名患者队列的 RT-PCR 分析先前确定的疾病复发风险的 RNA 生物标志物，并且还鉴定出了使用先前在另一组患者中使用 DNA 微阵列发现的高百分比的复发风险标志物，这表明这种 RNA-Seq 技术具有足够的精度和灵敏度用于生物标志物发现。在 136 名患者队列中，有 2000 多个 RNA 与乳腺癌复发风险强烈相关（FDR <10%）。其中许多是与疾病复发没有关联的内含子 RNA。与复发风险相关的一些 RNA 属于新的 RNA 网络。在对其他乳腺癌队列的全转录组 RNA-Seq 筛选中测试这些新关联的有效性将非常重要。
