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

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.

Preanalytical quality improvement: in quality we trust

“…The biggest problem is the extraction of nucleic acids, as they are often degraded, cross-linked to proteins and modified. Automated extraction is available and should henceforth be preferred [43] . Extracted material is usually well-suited for polymerase chain reaction (PCR), sequencing faces to DNA breaks and other DNA modifications [44] .…”

Evaluation of the association of PIK3CA mutations and PTEN loss with efficacy of trastuzumab therapy in metastatic breast cancer

“…Single nucleotide polymorphism (SNP) genotyping for PIK3CA mutations DNA was extracted from 182 FFPE whole tissue sections or macrodissected tissue fragments containing >70% tumor cells, using a fully automated isolation method based on silica-coated magnetic beads (Versant Tissue Preparation Reagents, Siemens Healthcare Diagnostics, Tarrytown, NY) in combination with a liquid handling robot, as previously described [21]. Mutation testing for PIK3CA E542K and E545K (exon Association of markers with HER2 status ER positivity was seen more frequently in HER2-negative patients (82% in HER2-negative vs. 59% in HER2-positive, p=0.001).…”

Comparison of different approaches for assessment of HER2 expression on protein and mRNA level: prediction of chemotherapy response in the neoadjuvant GeparTrio trial (NCT00544765)

“…RNA was isolated from formalin-fixed, paraffinembedded (FFPE) tumor tissue specimens employing a Siemens Healthcare Diagnostics proprietary experimental method based on magnetic beads (not commercially available) as previously described [8]. All tumor samples included in the study contained at least 30% tumor tissue, as evaluated by hematoxylin-eosin staining.…”

“…All tumor samples included in the study contained at least 30% tumor tissue, as evaluated by hematoxylin-eosin staining. RPL37A, a house keeping gene and HER2 mRNA expression were assessed by kinetic one-step RT-PCR on ABI7900 systems using primers and probes as described previously [8]. Forty amplification cycles were applied and the threshold (Ct) values of genes were determined by ABI PRISM software.…”

“…On the basis of this observation, we have developed a quantitative method for determination of HER2 mRNA expression in FFPE tissue [7,8]. The aim of this study was to compare different methods for HER2 testing and to identify the predictive value of HER2 expression for a pathological complete response to neoadjuvant anthracycline/taxane-based chemotherapy.…”