The methodological challenge in high-throughput profiling and quantifying microRNAs

Abstract

Background: MicroRNAs (miRNAs) play an essential role in various biological processes and signaling pathways through the regulation of gene expression and genome stability. Recent data indicated that the next-generation sequencing (NGS)-based high-throughput quantification of miRNAs from biofluids provided exciting possibilities for discovering biomarkers of various diseases and might help promote the development of the early diagnosis of cancer. However, the complex process of library construction for sequencing always introduces bias, which may twist the actual expression levels of miRNAs and reach misleading conclusions.

Results: We discussed the deviation issue in each step during constructing miRNA sequencing libraries and suggested many strategies to generate high-quality data by avoiding or minimizing bias. For example, improvement of adapter design (a blocking element away from the ligation end, a randomized fragment adjacent to the ligation junction and UMI) and optimization of ligation conditions (a high concentration of PEG 8000, reasonable incubation temperature and time, and the selection of ligase) in adapter ligation, high-quality input RNA samples, removal of adapter dimer (solid phase reverse immobilization (SPRI) magnetic bead, locked nucleic acid (LNA) oligonucleotide, and Phi29 DNA polymerase), PCR (linear amplification, touch-down PCR), and product purification are essential factors for achieving high-quality sequencing data. Moreover, we described several protocols that exhibit significant advantages using combinatorial optimization and commercially available low-input miRNA library preparation kits.

Conclusions: Overall, our work provides the basis for unbiased high-throughput quantification of miRNAs. These data will help achieve optimal design involving miRNA profiling and provide reliable guidance for clinical diagnosis and treatment by significantly increasing the credibility of potential biomarkers.

Author summary

Given the central importance of accurate quantification of miRNAs to molecular biology and clinical diagnosis, in this work, we reviewed recent findings on NGS-based quantification methods of miRNAs and discussed the possible deviations of each step. A series of optimization strategies were proposed to avoid or minimize such biases. In addition, combination optimization of various conditions during library preparation and eight commercially available low-input miRNA library preparation kits were described. Our work points out the problems in the existing library preparation process and summarizes possible optimization conditions that can be used for high-throughput profiling and quantifying microRNAs.

Graphical abstract

Keywords

microRNA / next-generation sequencing / library preparation / bias

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ACKNOWLEDGEMENTS

This work was supported by the National Science and Technology Major Project during the 13th 5-Year Plan Period (No. 2019ZX09721001-007-002).

COMPLIANCE WITH ETHICS GUIDELINES

The authors Mengya Chai, Xueyang Xiong, Huimin Wang and Lida Xu declare that they have no conflicts of interests.

This article is a review article and does not contain any studies with human or animal subjects performed by any of the authors.

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