Optimization of RNA Extraction from Formalin-Fixed Paraffin-Embedded Blocks for Targeted Next-Generation Sequencing.

Abstract

PurposeBreast cancer has a high prevalence in Korea. To achieve personalized therapy for breast cancer, long-term follow-up specimens are needed for next-generation sequencing (NGS) and multigene analysis. Formalin-fixed paraffin-embedded (FFPE) samples are easier to store than fresh frozen (FF) samples. The objective of this study was to optimize RNA extraction from FFPE blocks for NGS.MethodsRNA quality from FF and FFPE tissues (n=5), expected RNA amount per unit area, the relationship between archiving time and quantity/quality of FFPE-extracted RNA (n=14), differences in quantitative real-time polymerase chain reaction (qRT-PCR) and NGS results, and comparisons of both techniques with tissue processing at different institutions (n=96) were determined in this study.ResultsThe quality of RNA did not show any statistically significant difference between paired FF and FFPE specimens (p=0.49). Analysis of tumor cellularity gave an expected RNA amount of 33.25 ng/mm2. Archiving time affected RNA quality, showing a negative correlation with RNA integrity number and a positive correlation with threshold cycle. However, RNA from samples as old as 10 years showed a 100% success rate in qRT-PCR using short primers, showing that the effect of archiving time can be overcome by proper experiment design. NGS showed a higher success rate than qRT-PCR. Specimens from institution B (n=46), which were often stored in a refrigerator for more than 6 hours and fixed without slicing, showed lower success rates and worse results than specimens from the other institutes.ConclusionArchived FFPE tissues can be used to extract RNA for NGS if they are properly processed before fixation. The expected amount of RNA per unit size calculated in this study will be useful for other researchers.