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Direct electrophoretic microRNA preparation from clinical samples using nanofilter membrane
Kidan Lee1, Jae-Hyun Kang1, Hyun-Mi Kim2
1Department of Materials Science and Engineering, Seoul National University, Seoul, 08826, Republic of Korea.
Nano Convergence
|January 14, 2020
Summary
This study introduces a novel method using an electric field and nanofilter membrane to directly collect nucleic acids like DNA and RNA from biosamples. The technique simplifies nucleic acid preparation and shows promise for clinical applications, including liquid biopsies.
Area of Science:
- Biotechnology
- Molecular Biology
- Nanotechnology
Background:
- Traditional nucleic acid extraction methods can be complex and time-consuming.
- Efficient isolation of nucleic acids from biological samples is crucial for downstream molecular analyses.
- Developing simpler, faster, and effective nucleic acid preparation techniques is an ongoing need in diagnostics and research.
Purpose of the Study:
- To propose and validate a novel method for direct nucleic acid collection from biosamples using electrophoresis.
- To demonstrate the efficiency, yield, and purity of nucleic acids isolated by the proposed electrical method.
- To assess the applicability of this method for clinical diagnostics, specifically in liquid biopsies for hepatocellular carcinoma detection.
Main Methods:
- Fabrication of a nanofilter membrane from low-stress silicon nitride with precisely engineered nanopores using nanoimprint technology.
- Application of an electric field to drive the electrophoretic transport of negatively charged nucleic acids (DNA, RNA, miRNA) across the nanofilter membrane into a collection buffer.
- Validation using quantitative reverse transcription-polymerase chain reactions (qRT-PCR) to confirm the transport and quantify collected microRNA (miRNA).
- Testing the method with clinical samples (sera from hepatocellular carcinoma patients and healthy controls) for liquid biopsy applications.
Main Results:
- Successful direct collection of nucleic acids, including specific microRNA (hsa-mir-93-5p), via electrophoretic transport across a 200 nm pore-sized nanofilter membrane.
- Demonstrated consistency in collected miRNA quantity, system stability, and high yield and purity of the prepared nucleic acid samples.
- Validated the method's effectiveness and compatibility with downstream applications like qRT-PCR.
- Successfully applied the method to evaluate miRNA levels in clinical serum samples, demonstrating its potential for liquid biopsy.
Conclusions:
- The proposed electrical method offers a simple and efficient physical approach for nucleic acid preparation from biosamples.
- The system is stable, yields high-purity nucleic acids, and is compatible with essential downstream analyses like qRT-PCR.
- This technique shows significant potential for simplifying nucleic acid isolation in both research settings and clinical diagnostics, particularly for liquid biopsies.
