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

Journal of Clinical Bioinformatics

Anton Belousov¹  Stephanie Wessner¹  Martin Weisser¹  Manuela Poignée-Heger¹  Marlene Thomas¹ 

[1] Pharma Research and Early Development (pRED), Roche Diagnostics GmbH, TR-H, Bldg 231/206a, Nonnenwald 2, 82377 Penzberg, Germany

关键词: Microarray;    HER2;    Gene;    Breast cancer;    Biomarker;   

Others  :  802935
DOI  :  10.1186/2043-9113-3-10

 received in 2012-12-07, accepted in 2013-04-23,  发布年份 2013

【 摘 要 】

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.

【 授权许可】

   
2013 Thomas et al.; licensee BioMed Central Ltd.

【 图 表 】

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