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Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material
Published on: August 1, 2018
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A Programmable Automatic Cascade Machinery for Single-Molecule Profiling of Multiple Noncoding RNAs in Breast
Wen Li1, Wen-Jing Liu1, Jun Lu2
1School of Chemistry and Chemical Engineering, State Key Laboratory of Digital Medical Engineering, Southeast University, Nanjing 211189, China.
Analytical Chemistry
|February 11, 2025
Summary
We developed a new method for detecting multiple noncoding RNAs (ncRNAs) in cells and tissues. This approach enables sensitive, single-molecule profiling for potential cancer diagnostics.
Area of Science:
- Molecular Biology
- Biochemistry
- Genomics
Background:
- Noncoding RNAs (ncRNAs) play crucial roles in cancer development and progression.
- Detecting specific ncRNA expression patterns in cells and tissues is vital for cancer diagnosis.
- Accurate and sensitive methods are needed to profile low-abundance ncRNAs.
Purpose of the Study:
- To develop a programmable, automated system for single-molecule profiling of multiple ncRNAs.
- To achieve sensitive detection and quantification of specific ncRNAs for potential diagnostic applications.
- To demonstrate the feasibility of the method in distinguishing between healthy and cancerous tissues.
Main Methods:
- Development of a programmable automatic cascade machinery utilizing two successive amplification events.
- Conversion of target ncRNAs into abundant FAM/Cy5 fluorescent molecules.
- Single-molecule imaging for detecting piR-36026 (FAM) and DSCAM-AS1 (Cy5).
- Programming dumbbell probes for recognition of specific ncRNA sequences.
Main Results:
- Achieved a highly sensitive detection limit of 44.67 aM for piR-36026 and 45.71 aM for DSCAM-AS1.
- Enabled single-cell level measurement of endogenous piR-36026 and DSCAM-AS1.
- Demonstrated successful profiling of these ncRNAs in both healthy and breast cancer tissues.
- High efficiency and signal-to-noise ratio of the automatic cascade machinery.
Conclusions:
- The developed method provides sensitive, single-molecule profiling of multiple ncRNAs.
- The system shows significant potential for cancer diagnostics by analyzing ncRNA expression.
- Programmable nature allows extension to other cancer-related ncRNAs, highlighting clinical application prospects.
