High-throughput, Efficient, and Unbiased Capture of Small RNAs from Low-input Samples for Sequencing.

Abstract:

:MicroRNAs hold great promise as biomarkers of disease. However, there are few efficient and robust methods for measuring microRNAs from low input samples. Here, we develop a high-throughput sequencing protocol that efficiently captures small RNAs while minimizing inherent biases associated with library production. The protocol is based on early barcoding such that all downstream manipulations can be performed on a pool of many samples thereby reducing reagent usage and workload. We show that the optimization of adapter concentrations along with the addition of nucleotide modifications and random nucleotides increases the efficiency of small RNA capture. We further show, using unique molecular identifiers, that stochastic capture of low input RNA rather than PCR amplification influences the biased quantitation of intermediately and lowly expressed microRNAs. Our improved method allows the processing of tens to hundreds of samples simultaneously while retaining high efficiency quantitation of microRNAs in low input samples from tissues or bodily fluids.

journal_name

Sci Rep

journal_title

Scientific reports

authors

Belair CD,Hu T,Chu B,Freimer JW,Cooperberg MR,Blelloch RH

doi

10.1038/s41598-018-38458-7

subject

Has Abstract

pub_date

2019-02-19 00:00:00

pages

2262

issue

1

issn

2045-2322

pii

10.1038/s41598-018-38458-7

journal_volume

9

pub_type

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