Template-dependent multiple displacement amplification for profiling human circulating RNA

19Citations
Citations of this article
60Readers
Mendeley users who have this article in their library.

This article is free to access.

Abstract

Multiple displacement amplification (MDA) is widely used in whole-genome/transcriptome amplification. However, template-independent amplification (TIA) in MDA is a commonly observed phenomenon, particularly when using high concentrations of random hexamer primers and extended incubation times. Here, we demonstrate that the use of random pentamer primers with 5’ ends blocked by a C18 spacer results in MDA solely in a template-dependent manner, a technique we have named tdMDA. Together with an optimized procedure for the removal of residual genomic DNA during RNA extraction, tdMDA was used to profile circulating RNA from 0.2 mL of patient sera. In comparison to regular MDA, tdMDA demonstrated a lack of quantifiable DNA amplification in the negative control, a remarkable reduction of unmapped reads from Illumina sequencing (7 ± 10.9% versus 58.6 ± 39%, P = 0.006), and increased mapping rates of the serum transcriptome (26.9 ± 7.9% versus 5.8 ± 8.2%, P = 3.8 × 10-4). Transcriptome profiles could be used to separate patients with chronic hepatitis C virus (HCV) infection from those with HCV-associated hepatocellular carcinoma (HCC). We conclude that tdMDA should facilitate RNA-based liquid biopsy, as well as other genome studies with biological specimens having ultralow amounts of genetic material.

Cite

CITATION STYLE

APA

Wang, W., Ren, Y., Lu, Y., Xu, Y., Crosby, S. D., Di Bisceglie, A. M., & Fan, X. (2017). Template-dependent multiple displacement amplification for profiling human circulating RNA. BioTechniques, 63(1), 21–27. https://doi.org/10.2144/000114566

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Save time finding and organizing research with Mendeley

Sign up for free