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.

Large scale, robust, and accurate whole transcriptome profiling from clinical formalin-fixed paraffin-embedded samples

“…Jovanovic et al . 45 demonstrated that subtyping accuracy of FFPE and FF triple negative breast cancer (TNBC) samples increased with sequencing depth, and that samples with shorter archiving time (< 4 years) were subtyped more accurately, presumably due to reduced RNA degradation which is consistent with others investigators 46 . However, Jovanovic focused their analysis on genes that did not vary between platform type, tissue processing, and RNA isolation techniques, tacitly suggesting that certain transcripts were unreliable and should be excluded.…”

Eosinophils and Macrophages within the Th2-Induced Granuloma: Balancing Killing and Healing in a Tight Space

“…Bulk RNA sequencing has successfully been performed on RNA obtained from the granuloma of nematode-infected mice using laser capture microdissection (24). However, it is now possible to isolate DNA/RNA from formalin-fixed, paraffin-embedded tissue sections (173,174). This is a great step forward, since many historical samples can now be analyzed.…”

Heterogeneity at the invasion front of triple negative breast cancer cells

“…While there have been other reports studying spatial heterogeneity within patient samples, these studies utilized the use of formalin fixed primary tumor tissue for the identification of genomic markers 15,16 . These studies mainly focus on identifying genomic mutations as markers of invasion as the deleterious effects of formalin on RNA integrity has been well documented and although technological advances in processing has resulted in improvements in nucleic acid quality, time of fixation plays a crucial role in the process, making such approaches inconsistent when studying gene expression 17,18 . In this study, we adopted a different approach toward understanding the heterogeneity existing within TNBC, by using a previously reported fluidic device to enrich for live cells found within the invasion front of this disease subtype for the specific purpose of studying the gene expression profile associated with invasion 19 .…”