Application Note Abstract 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 paraffi n-embedded (FFPE) tissues are the only samples available. To work with these diffi cult 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 amplifi ed using a system in which a reverse transcription-generated cDNA library is subjected to limited PCR amplifi cation. 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 repli- cates. Principal component analysis of array signals demonstrated clear separa- tion 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 showed similar expression level changes.
Microarray-based gene expression analysis typically requires high-quality RNA from fresh frozen tissues. However, many studies rely on the vast archive of formalin-fixed paraffin-embedded (FFPE) tissue samples. This document details a specific protocol developed by Agilent to optimize microarray analysis for these difficult FFPE samples.
The study compares gene expression profiles derived from FFPE-derived RNA against matched fresh frozen samples using colon tumor and normal tissues. The workflow includes specialized RNA extraction, amplification via the TransPlex Whole Transcriptome Amplification system, labeling with the Universal Linkage System (ULS), hybridization to Agilent 4x44K microarrays, and data analysis using GeneSpring GX software.
| Microarray Type | Agilent Whole Human Genome 4x44K Microarrays |
|---|---|
| Probe Count | 41,000 unique in situ synthesized 60-mer oligonucleotide biological probes |
| Hybridization Duration | 17 hours |
The Universal Linkage System (ULS) was chosen because it is a direct labeling method independent of fragment length.
Both FFPE and frozen samples revealed more than 2200 genes with a fold-change greater than 2.