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Related Experiment Video

Updated: Mar 21, 2026

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens
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From RNA isolation to microarray analysis: Comparison of methods in FFPE tissues.

Nevin Belder1, Öznur Coskun2, Beyza Doganay Erdogan3

  • 1Ankara University, Biotechnology Institute, Ankara, Turkey.

Pathology, Research and Practice
|May 11, 2016
PubMed
Summary

Optimizing RNA extraction from FFPE tissues is crucial for cancer research. The Qiagen RNeasy FFPE kit with a modified deparaffinization step and the Ribo-SPIA protocol yield superior results for gene expression profiling.

Keywords:
FFPE tissuesGene expressionMicroarrayRNA isolation

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Area of Science:

  • Molecular Biology
  • Cancer Research
  • Genomics

Background:

  • Formalin-fixed paraffin-embedded (FFPE) tissues are vital for cancer research.
  • Challenges in FFPE sample processing include nucleic acid degradation and fragmentation.
  • Optimization of RNA extraction and microarray analysis is needed for FFPE samples.

Purpose of the Study:

  • To optimize RNA isolation and microarray analysis protocols for FFPE tissue samples.
  • To compare the efficacy of different RNA isolation kits and cDNA preparation methods.
  • To evaluate the impact of array platforms on gene expression profiling results.

Main Methods:

  • Modified deparaffinization protocol followed by RNA isolation using Trizol, Qiagen RNeasy FFPE, and Arcturus PicoPure kits.
  • Comparison of two cDNA/cRNA preparation and labeling protocols (Ribo-SPIA vs. 3' IVT).
  • Analysis using two microarray platforms (Affymetrix Human Genome U133 Plus 2.0 and U133_X3P).

Main Results:

  • The Qiagen RNeasy FFPE kit with the modified deparaffinization step yielded higher RNA quantity and quality.
  • The Ribo-SPIA protocol resulted in a significantly higher percentage of present calls compared to the 3' IVT system.
  • No significant differences in results were observed between the two array platforms.

Conclusions:

  • Optimized FFPE tissue processing enhances RNA isolation and microarray analysis.
  • The Qiagen RNeasy FFPE kit and Ribo-SPIA protocol are recommended for FFPE gene expression studies.
  • This optimization facilitates high-throughput transcriptional analysis from FFPE samples.