Whole-Genome Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded Tissue Samples
Open Access
- 3 December 2009
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 4 (12), e8162
- https://doi.org/10.1371/journal.pone.0008162
Abstract
We have developed a gene expression assay (Whole-Genome DASL®), capable of generating whole-genome gene expression profiles from degraded samples such as formalin-fixed, paraffin-embedded (FFPE) specimens. We demonstrated a similar level of sensitivity in gene detection between matched fresh-frozen (FF) and FFPE samples, with the number and overlap of probes detected in the FFPE samples being approximately 88% and 95% of that in the corresponding FF samples, respectively; 74% of the differentially expressed probes overlapped between the FF and FFPE pairs. The WG-DASL assay is also able to detect 1.3–1.5 and 1.5–2 -fold changes in intact and FFPE samples, respectively. The dynamic range for the assay is ∼3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R2 ∼0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R2∼0.86 and R2∼0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R2>0.98) with low intact RNA inputs ranging from 1 ng to 100 ng; reproducible expression profiles were also obtained with 250 pg total RNA (R2∼0.92), with ∼71% of the probes detected in 100 ng total RNA also detected at the 250 pg level. When FFPE samples were assayed, 1 ng total RNA yielded self-correlations of R2∼0.80, while still maintaining a correlation of R2∼0.75 with standard FFPE inputs (200 ng). Taken together, these results show that WG-DASL assay provides a reliable platform for genome-wide expression profiling in archived materials. It also possesses utility within clinical settings where only limited quantities of samples may be available (e.g. microdissected material) or when minimally invasive procedures are performed (e.g. biopsied specimens).Keywords
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