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. 2016 Aug 20;9(1):54.
doi: 10.1186/s12920-016-0215-4.

Reliable gene expression profiling of formalin-fixed paraffin-embedded breast cancer tissue (FFPE) using cDNA-mediated annealing, extension, selection, and ligation whole-genome (DASL WG) assay

Mahesh Iddawela  1   2   3   4   5   6 , Oscar M Rueda  7 , Marcus Klarqvist  7 , Stefan Graf  7 , Helena M Earl  8   9   10 , Carlos Caldas  7   8   9   10
Affiliations

Reliable gene expression profiling of formalin-fixed paraffin-embedded breast cancer tissue (FFPE) using cDNA-mediated annealing, extension, selection, and ligation whole-genome (DASL WG) assay

Mahesh Iddawela et al. BMC Med Genomics. .

Abstract

Background: 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 (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.

Method: RNA 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.

Results: 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 FF). There was a significant degree of correlation between replicates within the FF and FFPE sample sets (r (2) = 0.96-0.98) as well as between the two platforms (DASL vs. BeadArray r (2) = 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.

Conclusion: Both 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.

Keywords: Breast Cancer; FFPE; Molecular Markers; WG DASL.

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Figures

Fig. 1
Fig. 1
a Samples and the number of technical replicates used for the DASL WG and Illumina BeadArray analysis & (b) The number of probes detected above background from Fresh (F) and FFPE (P) samples in the DASL WG assay
Fig. 2
Fig. 2
Scatter plots showing correlations of gene expression between FF vs. FFPE samples assessed using the DASL WG assay, following selection of the best performing probes
Fig. 3
Fig. 3
Hierachial clustering of samples assessed using DASL WG assays. FFPE & FF samples assessed using the DASL WG assay clustered together in the majority of samples (note 99028 F is an outlier as it had very few probes detected)
Fig. 4
Fig. 4
Hierarchical clustering of FF and FFPE samples assessed using the DASL WG assay, and of FF samples assessed using the Illumina BeadArray. This shows significant clustering of FF and FFPE samples, regardless of whether the DASL WG or the BeadArray (indicated by number only) are used
See this image and copyright information in PMC

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