Clinical Chemistry
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Clinical Chemistry 55: 1995-2003, 2009. First published September 10, 2009; 10.1373/clinchem.2009.128215
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(Clinical Chemistry. 2009;55:1995-2003.)
© 2009 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

A High-Sensitivity, Medium-Density, and Target Amplification–Free Planar Waveguide Microarray System for Gene Expression Analysis of Formalin-Fixed and Paraffin-Embedded Tissue

Stephan Schwers1,a, Elke Reifenberger1, Mathias Gehrmann1, Alexandre Izmailov2 and Kerstin Bohmann1

1 Siemens Healthcare Diagnostics Products GmbH, Molecular Research Germany, Cologne, Germany; 2 Siemens Healthcare Diagnostics Ltd., Toronto, ON, Canada.

aAddress correspondence to this author at: Siemens Healthcare Diagnostics Products GmbH, Bldg. S19, Nattermannallee 1, 50829 Cologne, Germany. Fax +49-221-576-3621; e-mail stephan.schwers{at}siemens.com.

Background: Many microarray platforms and their associated assay chemistries do not work properly with RNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue samples, a feature that severely hampers the use of microarrays in oncology applications, for which FFPE tissue is the routine specimen. Furthermore, the limited sensitivity of most microarray platforms requires time-consuming and costly amplification reactions of the target RNA, which negatively affects clinical laboratory work flow.

Methods: We developed an approach for sensitively and reliably measuring mRNA abundances in FFPE tissue samples. This approach involves automated RNA extractions, direct hybridization of extracted RNA to immobilized capture probes, antibody-mediated labeling, and readout with an instrument applying the principle of planar waveguides (PWG). A 14-gene multiplex assay conducted with RNA isolated from 20 FFPE blocks was correlated to an analysis of the same with reverse-transcription quantitative real-time PCR (RT-qPCR).

Results: The assay sensitivity for gene expression analysis obtained for the PWG microarray platform was <10 fmol/L, eliminating the need for target preamplification. We observed a correlation coefficient of 0.87 to state-of-the-art RT-qPCR technology with RNA isolated from FFPE tissue, despite a compressed dynamic range for the PWG system (a 2.9-log dynamic range for PWG in our test system vs 5.0 logs for RT-qPCR). The precision of the PWG platform was comparable to RT-qPCR (Pearson correlation coefficient of 0.9851 for PWG vs 0.9896 for RT-qPCR) for technical replicates.

Conclusions: The presented PWG platform demonstrated excellent sensitivity and precision and is especially well suited for any application for which fast, simple, and robust multiplex assays of RNA in FFPE tissue are required.






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Copyright © 2009 by the American Association for Clinical Chemistry.