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Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations
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LINCATRA: Two-cycle method to amplify RNA for transcriptome analysis from formalin-fixed paraffin-embedded tissue.

Poorna Manasa Bhamidimarri1, Laila Salameh2, Amena Mahdami1

  • 1Research Institute for Medical and Health Sciences, University of Sharjah, Sharjah, United Arab Emirates.

Heliyon
|July 11, 2024
PubMed
Summary

Formalin Fixed Paraffin Embedded (FFPE) tissue RNA can now be analyzed for whole transcriptome analysis (WTA) using the new LINCATRA method. This technique amplifies degraded RNA from FFPE samples, enabling accurate gene expression analysis for disease research.

Keywords:
DegradedFormalin-fixed paraffin embeddedLinear amplificationRNA-SeqTranscriptomic analysis

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Area of Science:

  • Molecular pathology
  • Genomics
  • Biotechnology

Background:

  • Whole transcriptome analysis (WTA) of RNA from Formalin Fixed Paraffin Embedded (FFPE) tissues is crucial for understanding disease molecular pathology.
  • RNA extracted from FFPE tissues is often degraded or scarce, limiting gene expression analysis.
  • Existing linear amplification methods are primarily designed for cellular RNA.

Purpose of the Study:

  • To introduce LINCATRA (LINear amplifiCAtion of RNA for whole TRAnscriptome analysis), a novel method for amplifying RNA from FFPE tissues.
  • To demonstrate LINCATRA's ability to amplify short, fragmented RNA with high fidelity from minimal input amounts (as low as 5 ng).
  • To validate the utility of LINCATRA-amplified RNA for comprehensive gene expression analysis and disease mechanism elucidation.

Main Methods:

  • Development of LINCATRA, a random nonamer primer-based linear amplification method.
  • Application of a two-cycle amplification process to RNA extracted from FFPE tissues.
  • Comparison of whole transcriptome analysis (WTA) results from amplified and unamplified RNA using RNA-sequencing.

Main Results:

  • LINCATRA achieved significant RNA amplification (approximately 1000-fold, p < 0.0001) and improved mean read lengths (p < 0.05).
  • Increased mean read length positively correlated with on-target reads in both amplified and unamplified RNA-seq (Pearson's r = 0.71, p < 0.0001).
  • Gene expression analysis showed substantial overlap in differentially expressed genes between amplified and unamplified groups, validating the method.

Conclusions:

  • LINCATRA effectively amplifies degraded RNA from FFPE tissues, enabling robust whole transcriptome analysis.
  • The method enhances RNA quality and quantity, facilitating accurate gene expression profiling.
  • LINCATRA is a valuable tool for clinical molecular pathology, aiding in diagnostics and the study of disease mechanisms.