Gene Expression Microarray Analysis Of Archival FFPE Samples

By Anne Bergstom Lucas and Gary Lin, Agilent Technologies

Microarray-based gene expression hybridization is a powerful and proven technique for studying differential gene expression signatures in cancer. RNA extracted from freshly frozen tissues is optimal for microarray analysis; however, in many cases, formalin-fixed paraffin-embedded (FFPE) tissues are the only samples available. To work with these difficult samples, Agilent has developed an FFPE sample protocol optimized for use with Agilent gene expression microarrays. To test the effectiveness of this protocol, total RNA was extracted from a quadruplicate set of colon tumor (adenocarcinoma), and matched normal colon FFPE and fresh frozen tissues. RNA purity and quality were determined, and the RNA was amplified using a system in which a reverse transcription-generated cDNA library is subjected to limited PCR amplification. The resulting cDNA target was chemically labeled. Analysis of the microarray results revealed a reduced sensitivity of detection for the FFPE-derived RNA samples; however, the lists of detected genes showed high overlap between the FFPE and frozen samples. Comparison of gene expression signals demonstrated excellent assay reproducibility between the technical replicates. Principal component analysis of array signals demonstrated clear separation between the tumor and normal samples with grouping of replicate samples. Statistical analysis of the expression level variations between tumor and normal samples revealed that both the FFPE and frozen samples had more than 2200 genes with a fold-change greater than 2 with a corrected p-value of 0.05 or less. The FFPE and frozen samples showed good concordance of tumor vs. normal log2 ratios with few anti-correlated genes.