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Quantitative assessment of short amplicons in FFPE-derived long-chain RNA

Hui Kong1, Mengou Zhu2, Fengyun Cui1

  • 1Department of Pathology, Shanghai Medical College, Fudan University, Shanghai, 200032, China.

Scientific Reports
|November 29, 2014
PubMed

Insights

Analyzing long RNA in formalin-fixed paraffin-embedded (FFPE) tissues is challenging due to degradation. Using three short amplicons in quantitative RT-PCR enables accurate long RNA quantification in FFPE samples.

Area of Science:

  • Molecular biology
  • Genomics
  • Biotechnology

Background:

  • Formalin-fixed paraffin-embedded (FFPE) tissues are valuable for molecular medical research.
  • Degradation of long-chain RNA in FFPE tissues limits its analysis.
  • Accurate quantification of long RNA is crucial for understanding disease mechanisms.

Purpose of the Study:

  • To investigate the feasibility of quantifying long-chain RNA in FFPE tissues.
  • To compare the efficiency of short and long amplicons for RNA analysis in FFPE samples.
  • To develop a reliable method for long RNA quantification in FFPE tissues.

Main Methods:

  • Quantitative reverse-transcription PCR (qRT-PCR) was performed on FFPE and snap-frozen colorectal tissues.
  • Short (~60 bp) and long (~200 bp) amplicons were designed for 14 target RNAs (mRNAs and long noncoding RNAs).
  • A novel approach using 3 non-overlapping short amplicons was employed for FFPE samples.

Main Results:

  • Short amplicons showed more efficient amplification than long amplicons in both FFPE and snap-frozen tissues.
  • A single short amplicon demonstrated only 36% consistency in fold-change trends between FFPE and snap-frozen tissues.
  • Using 3 non-overlapping short amplicons achieved 100% concordance in fold-change trends for all target RNAs in FFPE tissues.

Conclusions:

  • Quantitative RT-PCR with 3 non-overlapping short amplicons enables accurate long-chain RNA analysis in FFPE tissues.
  • This method overcomes RNA degradation challenges in FFPE samples.
  • The findings support the use of FFPE tissues for comprehensive molecular profiling.

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