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Template-dependent multiple displacement amplification for profiling human circulating RNA.

Weihua Wang1,2, Yi Ren1, Yang Lu1

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|July 14, 2017
PubMed
Summary
This summary is machine-generated.

Template-dependent multiple displacement amplification (tdMDA) uses blocked pentamer primers to prevent unwanted DNA amplification. This method improves RNA profiling from patient sera for liquid biopsy and disease detection.

Keywords:
C18 spacerliquid biopsymultiple displacement amplification

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Multiple displacement amplification (MDA) is a common technique for whole-genome and whole-transcriptome amplification.
  • Template-independent amplification (TIA) is a known issue in MDA, especially with high primer concentrations and long incubation times.

Purpose of the Study:

  • To develop a template-dependent MDA (tdMDA) method using modified primers.
  • To optimize tdMDA for profiling circulating RNA from small patient serum samples.
  • To evaluate the performance of tdMDA compared to standard MDA for serum transcriptome analysis.

Main Methods:

  • Utilized random pentamer primers with 5´ ends blocked by a C18 spacer for tdMDA.
  • Implemented an optimized procedure for removing residual genomic DNA during RNA extraction.
  • Applied tdMDA to profile circulating RNA from 0.2 mL of patient sera and performed Illumina sequencing.

Main Results:

  • tdMDA showed no quantifiable DNA amplification in negative controls.
  • tdMDA significantly reduced unmapped sequencing reads (7 ± 10.9% vs. 58.6 ± 39%, P = 0.006).
  • tdMDA increased serum transcriptome mapping rates (26.9 ± 7.9% vs. 5.8 ± 8.2%, P = 3.8 × 10-4).
  • Serum transcriptome profiles generated by tdMDA differentiated patients with chronic hepatitis C virus (HCV) infection from those with HCV-associated hepatocellular carcinoma (HCC).

Conclusions:

  • tdMDA offers a reliable method for template-dependent amplification, minimizing artifacts.
  • Optimized tdMDA facilitates sensitive RNA-based liquid biopsy from minimal sample volumes.
  • This technique holds promise for various genomic studies involving scarce biological material.