Hybridization Protection Reaction for Sensitive and Robust Gene Expression Profiling of Clinical Formalin-Fixed Paraffin-Embedded Samples

Abstract

Abstract 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.