Abstract
BackgroundThe difficulties in using formalin-fixed and paraffin-embedded (FFPE) tumour specimens for molecular marker studies have hampered progress in translational cancer research. The cDNA-mediated, annealing, selection, extension, and ligation (DASL) assay is a platform for gene expression profiling from FFPE tissue and hence could allow analysis of large collections of tissue with associated clinical data from existing archives, therefore facilitating the development of novel biomarkers.MethodRNA isolated from matched fresh frozen (FF) and FFPE cancer specimens was profiled using both the DASL whole-genome (WG) platform, and Illumina BeadArray’s, and results were compared. Samples utilized were obtained from the breast cancer tumour bank held at the Cambridge University Hospitals NHS Foundation Trust.ResultsThe number of reliably detected probes was comparable between the DASL and BeadArray platforms, indicating that the source of RNA did not result in a significant difference in the detection rates (Mean probes- 17114 in FFPE & 17400 in FF). There was a significant degree of correlation between replicates within the FF and FFPE sample sets (r2 = 0.96–0.98) as well as between the two platforms (DASL vs. BeadArray r2 = range 0.83–0.89). Hierarchical clustering using the most informative probes showed that replicate and matched samples were grouped into the same sub-cluster, regardless of whether RNA was derived from FF or FFPE tissue.ConclusionBoth FF and FFPE material generated reproducible gene expression profiles, although there was more noise in profiles from FFPE specimens. We have shown that the DASL WG platform is suitable for profiling formalin-fixed paraffin-embedded samples, but robust bioinformatics analysis is required.
Highlights
The difficulties in using formalin-fixed and paraffin-embedded (FFPE) tumour specimens for molecular marker studies have hampered progress in translational cancer research
The number of reliably detected probes was comparable between the DASL and BeadArray platforms, indicating that the source of RNA did not result in a significant difference in the detection rates (Mean probes- 17114 in FFPE & 17400 in fresh frozen (FF))
Hierarchical clustering using the most informative probes showed that replicate and matched samples were grouped into the same sub-cluster, regardless of whether RNA was derived from FF or FFPE tissue
Summary
The difficulties in using formalin-fixed and paraffin-embedded (FFPE) tumour specimens for molecular marker studies have hampered progress in translational cancer research. The cDNA-mediated, annealing, selection, extension, and ligation assay (DASL®, Illumina, San Diego) is a platform that was developed for high-throughput gene expression analysis of partially degraded RNA. This assay initially assessed the expression of up to 1536 probes (a maximum of 512 genes, with three probes per gene) using 100–200 ng of degraded RNA [4]. The assay is designed to analyse 50 bp RNA fragments, and the sensitivity of the platform is enhanced by PCR amplification using common primers and by having 30 replicate beads per probe These arrays are assembled randomly, and the position of each probe is determined following manufacture, which reduces spatial effects [5]
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