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Extracting DNA and RNA from FFPE tissue blocks—Can we get there from here?

Posted by Abdul Ally on Jan 27, 2015 1:00:00 PM

tissue_sectioningThere is a wide range of pre-analytical factors affecting the uniformity of formalin-fixed paraffin embedded (FFPE) samples as potential sources of DNA and RNA.  Given these factors, we must ask ourselves, are FFPE samples reliable enough to advance progress toward precision medicine and the development of regenerative therapies?

FFPE tissue samples are widely available and offer, in addition to classic morphological study of tissues, the ability to harvest DNA, RNA and Proteins. Even more importantly, FFPE are accompanied by clinical data and their usefulness is further enhanced by still another factor—room temperature storage.

However, with regard to reliability, a review by Bass et al2 of pre-analytical factors that influenced molecular analyses of FFPE tissues identified 19 variables that have been investigated for potential effects, including decalcification, postmortem interval, specimen size, fixation temperature, duration of storage, fixative buffer, and others. The reviewers concluded that all 19 exerted an effect on downstream analyses of nucleic acids and protein analytes. They further listed 16 additional pre-analytical variables that have not yet been investigated.

FFPE tissues have substantial value, particularly for retrospective analyses that compare pathology with responses to treatment and clinical outcomes. In the past few years, the technology for extracting DNA and RNA has also dramatically improved. Formalin cross-linking has become less of a barrier, although fragmentation of the molecules continues to limit their usefulness.

There is no doubt that maintaining biological specimens in a frozen state is expensive. However, storing research materials in a manner that compromises their value for research is even more expensive, and conducting research on materials that are unsuited for the intended purpose can endanger an entire research program.

We are in an age of translational medicine and have a set of molecular tools that could not be imagined a generation ago, but we are not necessarily using these tools wisely. A 2014 review of current progress in translational research (in this case, specifically focused on oncology) by Doroshow and Kummer1 noted that despite well-known advances in the field—the HER2 and certain other predictive biomarkers easily come to mind—most attempts to identify and develop biomarkers using ‘omics’ technologies have not made the transition to clinical use. Doroshow and Kummer listed a number of issues that have slowed development, including inadequate attention to specimen preparation and quality, the definition of standard operating procedures, and lack of requirements for validation of assays.

Doroshow and Kummer noted that standardization of tissue collection and documentation of pre-analytical variables have become a priority in pathology laboratories. This is excellent news going forward. In the meantime, researchers should look closely at both the benefits and the consequences of archived FFPE tissue when the handling, fixation, processing, and storage parameters for a specimen are unknown.

Translational medicine requires high quality DNA and RNA for use in gene expression micro-arrays, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and analysis of DNA and RNA sequences. Given the current pre-analytical variability in tissue preservation and the limited shelf life3 of FFPE tissues (<10 years), in harvesting DNA and RNA of sufficient quality to accurately characterize mutations (for instance as predictors of molecular targeting therapeutics), we should consider extracting a portion of these tissues for DNA and RNA harvest sooner rather than later.

This is not a call to stop fixing and embedding tissues in paraffin for room temperature storage, but it is a call to make use of improved technologies for nucleic acid extraction. Further, emerging commercial technologies for the room temperature storage of nucleic acids—besides FTA paper—offer additional options for convenient and lower cost alternatives to both frozen and FFPE tissues. We should incorporate all these strategies, and depend less on FFPE tissues, to ensure the availability of critical precursors to successful drug discovery.

For related infromation about the optimal storage temperature for a wide array of samples, including DNA, RNA, and active proteins, download our infoposter below.

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 References:

1) Doroshow, J. H. & Kummar, S. (2014). Translational research in oncology—10 years of progress and future prospects. Nature Reviews | Clinical Oncology, Vol. 11, pp. 649–662.

2) Bass, B. P.; Engel, K. B.; Greytak, S. R. & Moore, H. M. (2014). A review of preanalytical factors affecting molecular, protein, and morphological analysis of formalin-fixed, paraffin-embedded (FFPE) tissue: How well do you know your FFPE specimen? Arch Pathol Lab Med, Vol. 138, pp. 1520–1530.

3) Chung J. Y. & Hewitt S. M. (2010). An optimized RNA extraction method from archival formalin-fixed paraffin-embedded tissue. Methods Mol Biol, Vol. 611, pp. 19 –27.