Gene Expression Profiling in Formalin-Fixed, Paraffin-Embedded Tissues Using the Whole-Genome DASL Assay

Abstract

Here, we provide a detailed technical description of a gene expression assay (Whole-Genome DASL (WG-DASL)), which not only enables whole-genome transcriptional profiling of degraded material, such as formalin-fixed, paraffin-embedded tissues, but is also capable of generating robust profiles with low input intact RNA. The WG-DASL assay combines target-specific annealing, extension, and ligation events followed by universal PCR and labeling steps to generate highly multiplexed Cy3-labeled products. These short products, which are single-stranded, are directly hybridized to a whole-genome expression BeadChip (HumanRef-8) containing probe content corresponding to ∼24 K RefSeq transcripts. After washing and imaging, fluorescence emissions are quantitatively recorded for each probe using high-resolution confocal scanners and imaging software. GenomeStudio software allows direct analysis of mRNA expression data and provides results in standard file formats that can be readily exported and analyzed with most standard gene expression analysis software programs. This technology is particularly useful for genome-wide expression profiling in degraded, archived material, including limited quantities of clinical samples, such as microdissected and biopsied materials.