Quantitative expression profiling in formalin-fixed paraffin-embedded samples by affymetrix microarrays.

Area of Science:

• Molecular Biology
• Genomics
• Bioinformatics

Background:

• Systematic characterization of microarray gene expression signal performance using degraded RNA from formalin-fixed, paraffin-embedded (FFPE) tissues versus intact RNA from fresh-frozen (FF) specimens is limited.
• Understanding RNA quality impact on gene expression profiling is crucial for utilizing archived samples.

Purpose of the Study:

• To systematically compare microarray gene expression signal performance between FFPE and FF tissues.
• To evaluate the impact of RNA degradation on probe-level and gene-level expression data.
• To establish guidelines for using FFPE samples in expression profiling studies.

Main Methods:

• RNA extraction and isolation from paired tumor and normal kidney, lung, and colon tissue specimens (FFPE and FF).
• Microarray signal dynamics evaluation at raw probe and probeset levels.
• Development of a contrast metric to compare FFPE and FF microarray signals.
• Comparison of gene-level summaries to determine expression profile overlap.

Main Results:

• RNA from FFPE tissues was more degraded and fragmented than from FF tissues, leading to a reduced dynamic range of expression signal.
• Probe performance was not uniform, declining significantly toward the 5' end of genes.
• Key differences between FFPE and FF signals were consistent across tissue types and enriched with ribosomal genes.

Conclusions:

• Archived FFPE samples are suitable for profiling expression signatures and assessing differential expression, similar to FF tissues.
• FFPE-derived expression data can be reliably used for discovery, validation, and clinical applications.
• The study provides essential guidelines for effective microarray expression profiling with FFPE material.