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Updated: May 28, 2025
Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material
Published on: August 1, 2018
A Programmable Automatic Cascade Machinery for Single-Molecule Profiling of Multiple Noncoding RNAs in Breast Tissues
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.
Abstract:
Noncoding RNAs (ncRNAs) are identified as critical regulatory molecules in tumorigenesis and progression. Investigating the expression patterns of multiple ncRNAs in living cells and tissues may facilitate the diagnosis of cancers. Herein, we develop a programmable automatic cascade machinery for single-molecule profiling of multiple ncRNAs. This method involves two successive amplification events that can convert extremely low-abundance target ncRNAs into abundant FAM/Cy5 molecules for the generation of amplified fluorescence signals. The subsequent single-molecule detection can identify piR-36026 with the FAM signal and DSCAM-AS1 with the Cy5 signal. Due to the high efficiency of automatic cascade machinery and the high signal-to-noise ratio of single-molecule imaging, this method can achieve sensitive detection of multiple ncRNAs with a detection limit of 44.67 aM for piR-36026 and 45.71 aM for DSCAM-AS1, and it can measure endogenous piR-36026 and DSCAM-AS1 at the single-cell level. Moreover, the profiling of piR-36026 and DSCAM-AS1 in healthy tissues and breast cancer tissues demonstrates the feasibility of the proposed method in cancer diagnostics. By programming the recognition sequences of dumbbell probes, this method can be extended to measure other cancer-related ncRNAs, with great prospects in clinical applications.
Insights
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.
