Abstract
The large archives of formalin-fixed paraffin-embedded (FFPE) tissue specimens that exist are a highly valuable source of sample material for molecular biological analysis, including gene expression profiling. However, current data on adverse effects of standard pathological practice on the usefulness of biomolecular analytes obtained from such archived specimens is largely anecdotal. Here, we present a systematic examination of the most relevant parameters for integrity and useability of RNA obtained from FFPE samples, including storage time and conditions, fixation time, and specimen size. The results are particularly relevant for any application relying on cDNA synthesis as an initial step of the procedure, such as RT-PCR, and microarray analysis.
Highlights
For preservation of tissue samples, formalin fixation followed by embedding in paraffin has been the method of choice for decades, mostly because this treatment maintains morphological features of the original tissue well
While chemical modification and crosslinking by formaldehyde is a direct consequence of the fixation process, fragmentation of RNA can result from a number of different sources: In the time period between sample acquisition and effective fixation, cellular processes and tissue autolysis can result in degradation of RNA, as well as other cellular components [7]
10 mm sections were cut for subsequent RNA isolation after 0, 1, 3, 6, and 12 months storage time, and RNA was purified at each time point from 1–4 sections using the RNeasy formalin-fixed paraffin-embedded (FFPE) kit
Summary
For preservation of tissue samples, formalin fixation followed by embedding in paraffin has been the method of choice for decades, mostly because this treatment maintains morphological features of the original tissue well. Relatively little is known about the individual impact of each of these different variables on the usefulness of samples for molecular applications, such as PCR. Such information can be helpful to predict potential usefulness of existing FFPE material for molecular studies, and perhaps for optimization of sample management for the future. While chemical modification and crosslinking by formaldehyde is a direct consequence of the fixation process, fragmentation of RNA can result from a number of different sources: In the time period between sample acquisition and effective fixation, cellular processes and tissue autolysis can result in degradation of RNA, as well as other cellular components [7]. Storage of sections can result in oxidation of RNA on the exposed surface, and staining procedures performed on sections may adversely affect RNA quality as well
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