Direct Quantification of Gene Expression in Homogenates of Formalin-Fixed, Paraffin-Embedded Tissues

Abstract

Formalin-fixed, paraffin-embedded (FFPE) tissues represent an important source of archival materials for gene expression profiling. We report here the development of a modified branch DNA assay that allows direct quantification of messenger RNA (mRNA) transcripts in homogenates from FFPE tissue sections without the need for RNA isolation and reverse transcription into cDNA. Formalin fixation essentially has no effect on the branch DNA assay, and RNA degradation only marginally reduces the signal by 2- to 3-fold. Under the same conditions, formalin fixation and RNA degradation greatly reduces real-time reverse transcription PCR (RT-PCR) efficiency, reducing signals by as much as 15- and 1400-fold, respectively. Although both technologies can generate biologically meaningful expression profiles from FFPE human lung tumor specimens, the branch DNA assay is more sensitive than real-time RT-PCR under the conditions tested. Our results therefore suggest that the branch DNA assay is an ideal tool for retrospective analysis of gene expression in archival tissues.

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“…However, formalin cross-links proteins and nucleic acids, which complicates their quantification [50,51]. RNA is significantly damaged before, during, and after FFPE procedure and is degraded into fragments below 200 bases [49,52,53] and chemically modified [51,54]. As a result, the efficiency of RNA extraction and reverse transcription are greatly reduced and in general, the measurement of RNA in FFPE tissues by polymerase chain reaction (PCR)-based methods is less reliable than in frozen tissues [54].…”