Dataset Information

An optimized workflow for improved gene expression profiling for formalin-fixed, paraffin-embedded tumor samples.

ABSTRACT:

Background

Whole genome microarray gene expression profiling is the 'gold standard' for the discovery of prognostic and predictive genetic markers for human cancers. However, suitable research material is lacking as most diagnostic samples are preserved as formalin-fixed, paraffin-embedded tissue (FFPET). We tested a new workflow and data analysis method optimized for use with FFPET samples.

Methods

Sixteen breast tumor samples were split into matched pairs and preserved as FFPET or fresh-frozen (FF). Total RNA was extracted and tested for yield and purity. RNA from FFPET samples was amplified using three different commercially available kits in parallel, and hybridized to Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays. The array probe set was optimized in silico to exclude misdesigned and misannotated probes.

Results

FFPET samples processed using the WT-Ovation™ FFPE System V2 (NuGEN) provided 80% specificity and 97% sensitivity compared with FF samples (assuming values of 100%). In addition, in silico probe set redesign improved sequence detection sensitivity and, thus, may rescue potentially significant small-magnitude gene expression changes that could otherwise be diluted by the overall probe set background.

Conclusion

In conclusion, our FFPET-optimized workflow enables the detection of more genes than previous, nonoptimized approaches, opening new possibilities for the discovery, validation, and clinical application of mRNA biomarkers in human diseases.

SUBMITTER: Thomas M

PROVIDER: S-EPMC3660273 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Publications

An optimized workflow for improved gene expression profiling for formalin-fixed, paraffin-embedded tumor samples.

Thomas Marlene M Poignée-Heger Manuela M Weisser Martin M Wessner Stephanie S Belousov Anton A

Journal of clinical bioinformatics 20130503 1

<h4>Background</h4>Whole genome microarray gene expression profiling is the 'gold standard' for the discovery of prognostic and predictive genetic markers for human cancers. However, suitable research material is lacking as most diagnostic samples are preserved as formalin-fixed, paraffin-embedded tissue (FFPET). We tested a new workflow and data analysis method optimized for use with FFPET samples.<h4>Methods</h4>Sixteen breast tumor samples were split into matched pairs and preserved as FFPET ...[more]

PMID: 23641797

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Project description:BackgroundWe 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.Methodology/principal findingsWe 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 approximately 3 logs. Comparing the WG-DASL assay with an in vitro transcription-based labeling method yielded fold-change correlations of R(2) approximately 0.83, while fold-change comparisons with quantitative RT-PCR assays yielded R(2) approximately 0.86 and R(2) approximately 0.55 for intact and FFPE samples, respectively. Additionally, the WG-DASL assay yielded high self-correlations (R(2)>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 (R(2) approximately 0.92), with approximately 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 R(2) approximately 0.80, while still maintaining a correlation of R(2) approximately 0.75 with standard FFPE inputs (200 ng).Conclusions/significanceTaken 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).

Project description:Endometriosis is an inflammatory pathology associated with a negative effect on life quality. Recently, this pathology was connected to ovarian cancer, in particular with endometrioid ovarian cancer. microRNAs (miRNAs) are a class of RNA transcripts ~19-22 nucleotides in length, the altered miRNA pattern being connected to pathological status. miRNAs are highly stable transcripts, and these can be assessed from formalin-fixed paraffin-embedded (FFPE) samples leading to the identification of miRNAs that could be developed as diagnostic and prognostic biomarkers, in particular those involved in malignant transformation. The aim of our study was to evaluate miRNA expression pattern in FFPE samples from endometriosis and ovarian cancer patients using PCR-array technology and also to compare the differential expression pattern in ovarian cancer versus endometriosis. For the PCR-array study, we have used nine macrodissected FFPE samples from endometriosis tissue, eight samples of ovarian cancers and five normal ovarian tissues. Quantitative real-time PCR (qRT-PCR) was used for data validation in a new patient cohort of 17 normal samples, 33 endometriosis samples and 28 ovarian cancer macrodissected FFPE samples. Considering 1.5-fold expression difference as a cut-off level and a P-value <0.05, we have identified four miRNAs being overexpressed in endometrial tissue, while in ovarian cancer 15 were differentially expressed (nine overexpressed and six downregulated). The expression level was confirmed by qRT-PCR for miR-93, miR-141, miR-155, miR-429, miR-200c, miR-205 and miR-492. Using the interpretative program Ingenuity Pathway Analysis revealed several deregulated pathways due to abnormal miRNA expression in endometriosis and ovarian cancer, which in turn is responsible for pathogenesis; this differential expression of miRNAs can be exploited as a therapeutic target. A higher number of altered miRNAs were detected in endometriosis versus ovarian cancer tissue, most of them being linked with epithelial-to-mesenchymal transition.

Dataset Information

An optimized workflow for improved gene expression profiling for formalin-fixed, paraffin-embedded tumor samples.

Background

Methods

Results

Conclusion

Publications

An optimized workflow for improved gene expression profiling for formalin-fixed, paraffin-embedded tumor samples.

Similar Datasets

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