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Quantifying mRNA in Highly Degraded Fixed Tissues by Nanostring Technology: A Comparative Study.
Eros Azzalini1, Barbara Di Stefano1, Vincenzo Canzonieri1,2
1Department of Medical Sciences, University of Trieste, 34149 Trieste, Italy.
Methods and Protocols
|May 28, 2024
Summary
nCounter Nanostring technology effectively quantifies mRNA in archival tissues, outperforming qPCR and ddPCR for both formalin-fixed and Bouin
Area of Science:
- Molecular biology
- Translational research
- Archival tissue analysis
Background:
- Archival tissues are crucial for molecular analysis but suffer biomolecule degradation.
- Formalin-fixed and paraffin-embedded (FFPE) tissues are standard, but Bouin's solution was also historically used.
- Investigating RNA integrity in historically processed tissues is vital for research.
Purpose of the Study:
- To assess nCounter Nanostring hybridization for quantifying mRNA in degraded Bouin's-fixed and paraffin-embedded (BFPE) tissues.
- To compare Nanostring performance against quantitative PCR (qPCR) and digital droplet PCR (ddPCR).
- To evaluate RNA quantification feasibility in FFPE and BFPE samples.
Main Methods:
- Analysis of 16 paraffin-embedded tissue blocks (8 FFPE, 8 BFPE).
- Application of Nanostring technology to 300 ng RNA samples.
- RNA retrotranscription and analysis via qPCR and ddPCR using 360 ng templates.
Main Results:
- Nanostring technology demonstrated superior performance in detecting target mRNA in both FFPE and BFPE samples compared to qPCR and ddPCR.
- The study confirmed Nanostring's effectiveness even with highly degraded RNA templates.
- RNA template degradation remains a limitation, potentially impacting gene expression analysis outcomes.
Conclusions:
- nCounter Nanostring is a powerful tool for mRNA quantification in archival tissues, including historically processed BFPE samples.
- While Nanostring offers advantages, researchers must remain aware of RNA degradation's impact on gene expression data.
- This study validates Nanostring for translational research utilizing diverse archival tissue specimens.
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