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Updated: Aug 1, 2026
Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations
Published on: August 4, 2016
Ultra-sensitive molecular detection of gene fusions from RNA using ASPYRE
Eleanor R Gray1, Justyna M Mordaka1, Efthimia R Christoforou1
1Biofidelity Ltd, 330 Cambridge Science Park, Milton road, CB4 0WN, Cambridge, England.
Background:
RNA is a critical analyte for unambiguous detection of actionable mutations used to guide treatment decisions in oncology. Currently available methods for gene fusion detection include molecular or antibody-based assays, which suffer from either being limited to single-gene targeting, lack of sensitivity, or long turnaround time. The sensitivity and predictive value of next generation sequencing DNA-based assays to detect fusions by sequencing intronic regions is variable, due to the extensive size of introns. The required depth of sequencing and input nucleic acid required can be prohibitive; in addition it is not certain that predicted gene fusions are actually expressed.
Results:
Herein we describe a method based on pyrophosphorolysis to include detection of gene fusions from RNA, with identical assay steps and conditions to detect somatic mutations in DNA [1], permitting concurrent assessment of DNA and RNA in a single instrument run.
Conclusion:
The limit of detection was under 6 molecules/ 6 µL target volume. The workflow and instrumentation required are akin to PCR assays, and the entire assay from extracted nucleic acid to sample analysis can be completed within a single day.
Insights
This study introduces a novel RNA-based method for detecting gene fusions, enabling concurrent DNA and RNA analysis. The assay offers high sensitivity and rapid results, improving oncologic mutation detection.
Area of Science:
- Molecular Biology
- Oncology Diagnostics
- Genomics
Background:
- RNA analysis is crucial for identifying actionable mutations in oncology, guiding treatment decisions.
- Existing gene fusion detection methods have limitations including single-gene targeting, low sensitivity, and long turnaround times.
- Next-generation sequencing DNA-based assays for fusion detection have variable sensitivity due to large intron sizes and may not confirm RNA expression.
Purpose of the Study:
- To develop a novel RNA-based method for gene fusion detection.
- To enable concurrent assessment of DNA and RNA in a single assay run.
- To improve upon the limitations of current gene fusion detection techniques.
Main Methods:
- A pyrophosphorolysis-based method was developed for detecting gene fusions directly from RNA.
- The assay utilizes identical steps and conditions for both DNA and RNA analysis.
- The workflow is designed to be similar to standard PCR assays.
Main Results:
- The method allows for the simultaneous detection of gene fusions from RNA alongside somatic mutations in DNA.
- Achieved a limit of detection below 6 molecules per 6 µL of target volume.
- The entire assay process, from nucleic acid extraction to sample analysis, can be completed within a single day.
Conclusions:
- The developed pyrophosphorolysis-based assay provides a sensitive and rapid method for RNA-based gene fusion detection.
- This approach permits concurrent DNA and RNA analysis, streamlining oncologic diagnostics.
- The assay's efficiency and speed offer a significant advancement over existing methods, with potential for faster clinical decision-making.
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