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Updated: Sep 5, 2025
Exosomal miRNA Analysis in Non-small Cell Lung Cancer NSCLC Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool
Published on: May 27, 2016
[Specific amplification of plasma exosome miRNA in cancer patients for construction of cDNA library]
Na Wang1, Fei He1, Hongwei Yu1
1College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China.
Abstract:
Plasma exosome microRNAs (miRNAs) are closely related with the occurrence, diagnosis, and treatment of cancers. However, the underlying molecular mechanisms remain unclear. We herein investigated the solution for tackling the unspecific amplification of plasma exosome microRNAs from cancer patients during the construction of its cDNA library. For the restriction enzyme digesting method, the primers were degraded by exonuclease T (EXOT) and phi29 DNA polymerase. For the magnetic bead separation method, the templates and primers were separated through the DNA binding beads. The separation effects of magnetic beads were detected by agarose gel electrophoresis and modified polyacrylamide gel electrophoresis. The levels of plasma exosome miRNAs from cancer patients and various primers were assayed by RT-qPCR. The results indicated that the unspecific amplification stemmed from USR5SR. EXOT and phi29 DNA polymerase could degrade USR5SR, but the templates were also degraded simultaneously. Regarding the magnetic bead separation method, the best effect was achieved via precipitation of primer fragments by 9% PEG and precipitation of templates by 15% PEG. In conclusion, the magnetic bead separation method efficiently circumvented the unspecific amplification during the construction of cDNA library, and therefore led to the successful construction of cDNA library from plasma exosome miRNA of cancer patients and 293T cells.
Insights
This study addresses unspecific amplification in plasma exosome microRNA (miRNA) cDNA library construction. A magnetic bead separation method effectively prevents this issue, enabling successful library creation for cancer research.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Plasma exosome microRNAs (miRNAs) are crucial biomarkers for cancer occurrence, diagnosis, and treatment.
- The molecular mechanisms linking exosome miRNAs to cancer are not fully understood.
- Unspecific amplification during cDNA library construction hinders accurate miRNA analysis.
Purpose of the Study:
- To investigate and resolve the problem of unspecific amplification in plasma exosome miRNA cDNA library construction.
- To compare enzymatic degradation with magnetic bead separation for eliminating unwanted amplification.
Main Methods:
- Investigated enzymatic degradation using exonuclease T (EXOT) and phi29 DNA polymerase.
- Developed and tested a magnetic bead separation method to isolate templates and primers.
- Utilized agarose gel electrophoresis, polyacrylamide gel electrophoresis, and RT-qPCR to assess separation efficiency and miRNA levels.
Main Results:
- Identified USR5SR as the source of unspecific amplification.
- Enzymatic degradation degraded USR5SR but also degraded target templates.
- Optimized magnetic bead separation using specific polyethylene glycol (PEG) concentrations for primer and template precipitation.
Conclusions:
- The magnetic bead separation method successfully prevents unspecific amplification in plasma exosome miRNA cDNA library construction.
- This method facilitates the successful creation of cDNA libraries from plasma exosome miRNAs of cancer patients and cell lines.
- Enables more reliable downstream analysis of exosome miRNA biomarkers in cancer.
