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Accurate data processing improves the reliability of Affymetrix gene expression profiles from FFPE samples.

Maurizio Callari1, Antonio Lembo2, Giampaolo Bianchini3

  • 1Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.

Plos One
|February 4, 2014
PubMed
Summary

Gene expression analysis from formalin-fixed paraffin-embedded (FFPE) tumor samples can be improved. Optimized data processing using alternative Chip Description Files (CDFs) and fRMA normalization enhances FFPE data reliability.

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Area of Science:

  • Molecular Biology
  • Bioinformatics
  • Cancer Research

Background:

  • Formalin-fixed paraffin-embedded (FFPE) tissues are standard clinical archives, valuable for biomarker development.
  • Gene expression profiling from FFPE samples using Affymetrix microarrays is feasible but shows discrepancies with fresh-frozen tissues.
  • Standard data processing methods yield inconsistent results from FFPE samples, necessitating improved analytical strategies.

Purpose of the Study:

  • To identify and validate optimized data processing methods for gene expression analysis from FFPE tumor specimens.
  • To improve the reliability and consistency of gene expression data derived from FFPE tissues compared to fresh-frozen samples.
  • To provide publicly available tools for implementing improved FFPE data processing.

Main Methods:

  • Comparison of various normalization methods and custom Chip Description Files (CDFs) on a matched frozen and FFPE gene expression dataset.
  • Application of fRMA normalization in conjunction with alternative CDFs.
  • Validation of the optimized processing pipeline using two independent FFPE datasets.

Main Results:

  • The use of alternative CDFs combined with fRMA normalization significantly enhanced correlations between frozen and FFPE samples and probesets.
  • Improved agreement in differential expression analysis across tumor subtypes was observed.
  • The optimized data processing strategy demonstrated robust performance and reproducibility across independent validation datasets.

Conclusions:

  • Appropriate data processing significantly enhances the reliability of gene expression data obtained from FFPE tissues using the Affymetrix platform.
  • The developed methods and publicly available tools offer a solution for consistent gene expression analysis from archival FFPE samples.
  • This approach facilitates more accurate biomarker discovery and validation from routinely collected clinical specimens.