Optimizing RNA Extraction Methods for High Throughput Transcriptome Sequencing of Fresh Frozen and Formalin-Fixed Paraffin-Embedded Cardiac Tissues Specimens

In this study, the efficiency of four RNA extraction methods was compared on 23 FFPE cardiac tissue specimens. The Qiagen AllPrep DNA/RNA FFPE kit (Method QP), Qiagen AllPrep DNA/RNA FFPE kit, with protocol modification on the ethanol wash step after deparaffinization (Method QE), CELLDATA RNAstorm 2.0 FFPE RNA Extraction kit (Method BP) and CELLDATA RNAstorm 2.0 FFPE RNA Extraction Kit with protocol modifications on the lysis step (Method BL). In comparing RNA quality metrics across FFPE RNA extract, nucleic acids extracted with Method QE and QP had the highest RNA yield. However, Method QE outperformed Method QP as more extract from Method QE had DV 200 values above 30%. Both method BL and BP produced a similar range of RNA purity and yield but more extract from Method BL had DV 200 values above 30% compared to Method BP. When accessing distribution value, Method BL outperformed Methods BP, QE, and QP as more extracts from Method BL had DV 200 values above 30% (16/23 samples) compared to other methods (PDV200<0.001; Kruskal-Wallis). However, method QE outperformed other methods in terms of RNA yield. The sequencing performance of RNA extracts from Method QE and Method BL was further tested on 8 matching samples with high RNA yield and high DV200 value respectively. RNA extracts from Method QE which yielded the highest RNA quantity among all methods exhibited comparable sequencing performance to extract obtained through Method BL, which yielded extract with high DV200 value. This study suggests that the DV200 and RNA yield are both reliable pre-analytic metrics in determining a suitable method for successful transcriptome sequencing of FFPE samples and have important implications for future studies exploring transcriptome sequencing of FFPE cardiac specimens. To generate quality transcriptome sequences for the identification of biomarkers predicting mortality in cardiac disease, we sought to generate a reliable and reproducible RNA extraction method. Four nucleic acid extraction methods (four methods) were compared on 23 FFPE cardiac tissue specimens. The RNA yield, RNA integrity, as reflected by the distribution value 200 (DV 200); and RNA purity, as reflected by the 260/280 and the 260/230 nm absorbance ratios, were determined. RNA sequencing was performed by deploying the Illumina Stranded Total RNA Ligation protocol on FFPE RNA extract. Bioinformatic analysis was performed to access transcriptome at gene and transcript level.