Abstract
Formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources commonly used in pathology. However, formalin fixation modifies nucleic acids challenging the isolation of high-quality RNA for genetic profiling. Here, we assessed feasibility and reliability of microarray studies analysing transcriptome data from fresh, fresh-frozen (FF) and FFPE tissues. We show that reproducible microarray data can be generated from only 2 ng FFPE-derived RNA. For RNA quality assessment, fragment size distribution (DV200) and qPCR proved most suitable. During RNA isolation, extending tissue lysis time to 10 hours reduced high-molecular-weight species, while additional incubation at 70 °C markedly increased RNA yields. Since FF- and FFPE-derived microarrays constitute different data entities, we used indirect measures to investigate gene signal variation and relative gene expression. Whole-genome analyses revealed high concordance rates, while reviewing on single-genes basis showed higher data variation in FFPE than FF arrays. Using an experimental model, gene set enrichment analysis (GSEA) of FFPE-derived microarrays and fresh tissue-derived RNA-Seq datasets yielded similarly affected pathways confirming the applicability of FFPE tissue in global gene expression analysis. Our study provides a workflow comprising RNA isolation, quality assessment and microarray profiling using minimal RNA input, thus enabling hypothesis-generating pathway analyses from limited amounts of precious, pathologically significant FFPE tissues.
Highlights
Pathological archives contain vast amounts of formalin-fixed paraffin-embedded (FFPE) specimen from routinely collected biopsies and autopsy tissue as well as precious material derived from rare disease cases or such with exceptional pathogenesis[1,2]
They passed functional integrity testing by yielding sufficient amounts of PCR products for selected reference genes (Fig. 3). To keep this transcript analysis as uniform and unbiased as possible, we used a mix of oligo(dT) and random hexamer primers for reverse transcription and primer sets annealing near the 5′- and 3′-end of the mRNA for qPCR (Table 1)
The same amounts of input were used for reverse transcription, fresh frozen (FF) samples resulted in smaller Cq values than FFPE samples for the tested reference genes indicating more PCR products (Fig. 3c,d)
Summary
Pathological archives contain vast amounts of formalin-fixed paraffin-embedded (FFPE) specimen from routinely collected biopsies and autopsy tissue as well as precious material derived from rare disease cases or such with exceptional pathogenesis[1,2]. Formaldehyde fixation (using formalin or paraformaldehyde) excellently preserves cellular structures and tissue morphologies by introducing inter- and intramolecular cross-links between side chains of amino acids[3,4], thereby maintaining secondary and tertiary structures of proteins[5,6]. In addition to fixation methods[8,10], studies have identified tissue fixation time, specimen size, and tissue storage conditions as important factors influencing RNA quality[11,12]. The choice of tissue lysis and isolation kits influences yield and quality of isolated RNA fragments[13,14,15,16]. Together with advanced transcription kits designed for low input derived from microdissected tissue, this opens up greater opportunities for basic and clinically-related research
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