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gene expression profiling from FFPE

Thursday 18 March 2004

Formalin-fixed, paraffin-embedded (FFPE) tissues represent an invaluable resource for cancer research, as they are the most widely available material for which patient outcomes are known.

There were over 300 million archived cancer tissue samples in the United States in 1999, with more samples accumulating at a rate of over 20 million per year.

The ability to perform gene expression profiling in these samples will enable both prospective and retrospective studies, and should greatly facilitate research in correlating expression profiles with clinical outcomes.

However, formalin fixation is known to render adenosine residues particularly prone to chemical modifications such as methylene dimerization and mono-methylolation and generate degraded RNA fragments (up to 50% of which may not contain a poly-A tract).

The degradation and chemical modification of RNA during tissue fixation and storage present challenges when applying conventional microarray technologies.


To overcome the technical limitations to microarray-based analyses of FFPE samples, a sensitive and reproducible gene expression profiling assay, DASL (cDNA-mediated annealing, selection, extension and ligation), have been developed for parallel analysis of hundreds of genes with highly degraded RNA samples. (19997620)

The DASL assay incorporates random priming during cDNA synthesis, and therefore does not depend solely on poly-A/oligo-dT based priming.

In addition, the assay requires a relatively short target sequence of about 50 nucleotides for query oligonucleotide annealing; thus, it can effectively quantify degraded RNA samples.

While most other technologies rely on multiple rounds of random priming for sample amplification and labeling, DASL only generates first strand cDNA that minimizes variation that arises during the random priming. (19997620)

This technology has been successfully used to profile a variety of archived FFPE tumor samples, some of which have been in storage for as long as 24 years and for which, in many cases, little or no tissue handling and fixation details are known, including colon, breast, lung, prostate, bladder, and liver cancer. (19997620)

While all of these applications clearly demonstrated the utility of the technology for identification and validation of tissue and cancer-specific markers, the DASL assay was limited by the number of genes that could be profiled simultaneously (up to 1536 targets). (19997620)


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