A high-sensitivity, medium-density, and target amplification-free planar waveguide microarray system for gene expression analysis of formalin-fixed and paraffin-embedded tissue.
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.
Schwers
Stephan
S
Siemens Healthcare Diagnostics Products GmbH, Molecular Research Germany, Bldg. S19, Nattermannallee 1, 50829 Cologne, Germany. stephan.schwers@siemens.com
Reifenberger
Elke
E
Gehrmann
Mathias
M
Izmailov
Alexandre
A
Bohmann
Kerstin
K
D001943:Breast Neoplasms / Q000235:genetics*; D005260:Female; D005557:Formaldehyde; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000191:economics / Q000295:instrumentation*; D016612:Paraffin Embedding; D012333:RNA, Messenger / Q000032:analysis*; D012680:Sensitivity and Specificity; D013997:Time Factors; D016707:Tissue Fixation
D012333:RNA, Messenger; D005557:Formaldehyde