RNA extraction method impacts quality metrics and sequencing results in FFPE samples

Archived formalin-fixed, paraffin-embedded (FFPE) tissue samples are being increasingly used in molecular cancer research. Compared with fresh-frozen tissue, the nucleic acid analysis of FFPE tissue is technically more challenging. Researchers at Ghent University aimed to compare the impact of 3 different RNA extraction methods on yield, quality, and sequencing-based gene expression results in FFPE samples.

RNA extraction was performed in 16 FFPE tumor specimens from patients with diffuse large B-cell lymphoma and in reference FFPE material from microsatellite-stable and microsatellite-instable cell lines (3 replicates each) using 2 silica-based procedures (A, miRNeasy FFPE; C, iCatcher FFPE Tissue RNA) and 1 isotachophoresis-based procedure (B, Ionic FFPE to Pure RNA). The RNA yield; RNA integrity, as reflected by the distribution value 200; and RNA purity, as reflected by the 260/280 and the 260/230 nm absorbance ratios, were determined. The RNA was sequenced on the NovaSeq 6000 instrument using the TruSeq RNA Exome and SMARTer Stranded Total RNA-Seq Pico v3 library preparations kits. These results highlight the impact of RNA extraction methodology on both preanalytical and sequencing-based gene expression results.

Overall, methods B and C outperformed method A because these showed significantly higher fractions of uniquely mapped reads, an increased number of detectable genes, a lower fraction of duplicated reads, and better representation of the B-cell receptor repertoire. Differences among the extraction methods were generally more explicit for the total RNA sequencing method than for the exome-capture sequencing method. Importantly, the predicative value of quality metrics varies among extraction kits, and caution should be applied when comparing and interpreting results obtained using different methods.

Fraction of uniquely mapped reads per RNA extraction method and library preparation in diffuse large B-cell lymphoma (n = 16) and microsatellite-stable or microsatellite-instable formalin-fixed, paraffin-embedded samples (n = 6). DLBCL, diffuse large B-cell lymphoma; MSI, microsatellite-instable; MSS, microsatellite-stable.

Decruyenaere P, Verniers K, Poma-Soto F, Van Dorpe J, Offner F, Vandesompele J. (2023) RNA Extraction Method Impacts Quality Metrics and Sequencing Results in Formalin-Fixed, Paraffin-Embedded Tissue Samples. Lab Invest 103(2):100027. [article]

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