BACKGROUND RNA profiling of formalin-fixed paraffin-embedded (FFPE) tumor tissues for the molecular diagnostics of disease prognosis or treatment response is often irreproducible and limited to a handful of biomarkers. This has led to an unmet need for robust multiplexed assays that can profile several RNA biomarkers of interest using a limited amount of specimen. Here, we describe hybridization protection reaction (HPR), which is a novel RNA profiling approach with high reproducibility. METHODS HPR assays were designed for multiple genes, including 10 radiosensitivity-associated genes, and compared with TaqMan assays. Performance was tested with synthetic RNA fragments, and the ability to analyze RNA was investigated in FPPE samples from 20 normal lung tissues, 40 lung cancer, and 30 esophageal cancer biopsies. RESULTS Experiments performed on 3 synthetic RNA fragments demonstrated a linear dynamic range of over 1000-fold with a replicate correlation coefficient of 0.99 and high analytical sensitivity between 3.2 to 10 000 pM. Comparison of HPR with standard quantitative reverse transcription polymerase chain reaction on FFPE specimens shows nonsignificant differences with > 99% confidence interval between 2 assays in transcript profiling of 91.7% of test transcripts. In addition, HPR was effectively applied to quantify transcript levels of 10 radiosensitivity-associated genes. CONCLUSIONS Overall, HPR is an alternative approach for RNA profiling with high sensitivity, reproducibility, robustness, and capability for molecular diagnostics in FFPE tumor biopsy specimens of lung and esophageal cancer.

中文翻译：

---

杂交保护反应，用于临床福尔马林固定石蜡包埋样品的敏感且稳健的基因表达谱分析。

背景技术用于疾病预后或治疗反应的分子诊断的福尔马林固定石蜡包埋（FFPE）肿瘤组织的RNA分析通常是不可重复的并且仅限于少数生物标志物。这导致对强大的多重检测的需求未得到满足，该检测可以使用有限数量的样本来分析多种感兴趣的 RNA 生物标志物。在这里，我们描述了杂交保护反应（HPR），这是一种具有高重复性的新型RNA分析方法。方法 HPR 检测针对多个基因（包括 10 个放射敏感性相关基因）设计，并与 TaqMan 检测进行比较。使用合成 RNA 片段测试了性能，并在来自 20 个正常肺组织、40 个肺癌和 30 个食道癌活检的 FPPE 样本中研究了 RNA 分析能力。结果 对 3 个合成 RNA 片段进行的实验表明，线性动态范围超过 1000 倍，重复相关系数为 0.99，分析灵敏度在 3.2 至 10 000 pM 之间。HPR 与标准定量逆转录聚合酶链式反应在 FFPE 样本上的比较显示，在 91.7% 测试转录本的转录本分析中，2 次检测之间存在 > 99% 置信区间的非显着差异。此外，HPR 还被有效地应用于量化 10 个放射敏感性相关基因的转录水平。结论 总体而言，HPR 是一种 RNA 分析的替代方法，具有高灵敏度、可重复性、稳健性，并且能够对肺癌和食道癌的 FFPE 肿瘤活检标本进行分子诊断。