RNA Extraction from Archival Formalin-Fixed Paraffin-Embedded Tissue: A Comparison of Manual, Semiautomated, and Fully Automated Purification Methods

Abstract

Abstract Background: Formalin-fixed paraffin-embedded (FFPE) tumor material represents a valuable resource for the analysis of RNA-based biomarkers, both in research laboratories and in routine clinical testing. A robust and automated RNA-extraction method with a high sample throughput is required. Methods: We evaluated extraction performance for 4 silica-based RNA-extraction protocols: (a) a fully automated, bead-based RNA-isolation procedure; (b) its manual counterpart; (c) a semiautomated bead-based extraction system; and (d) a manual column-based extraction kit. RNA from 360 sections (90 sections per extraction method) of 30 FFPE tumor blocks up to 20 years of age was purified and analyzed by quantitative reverse-transcription PCR for ESR1 (estrogen receptor 1), PGR (progesterone receptor), ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian)], and RPL37A (ribosomal protein L37a). Results: The semiautomated protocol gave the best yield. The 3 bead-based methods showed good across-method correlations in both yield and relative mRNA amounts (r = 0.86–0.95 and 0.98, respectively). In contrast, correlations between any of the bead-based methods and the manual column-based method were worse (r = 0.77–0.95 and 0.96, respectively). The fully automated method showed the lowest variation from section to section (root mean square error, 0.32–0.35 Cq, where Cq is the quantification cycle) and required the least hands-on time (1 h). Conclusions: The fully automated RNA-purification method showed the best reproducibility in gene expression analyses of neighboring sections of tissue blocks between 3 and 20 years of age and required the least overall and hands-on times. This method appears well suited for high-throughput RNA analyses in both routine clinical testing and translational research studies with archived FFPE material.