Messenger RNA capture sequencing of extracellular RNA from human biofluids using a comprehensive set of spike-in controls

Eva Hulstaert; Anneleen Decock; Annelien Morlion; Celine Everaert; Kimberly Verniers; Justine Nuytens; Nele Nijs; Gary P Schroth; Scott Kuersten; Stephen M Gross; Pieter Mestdagh; Jo Vandesompele

doi:10.1016/j.xpro.2021.100475

Messenger RNA capture sequencing of extracellular RNA from human biofluids using a comprehensive set of spike-in controls

STAR Protoc. 2021 Apr 14;2(2):100475. doi: 10.1016/j.xpro.2021.100475. eCollection 2021 Jun 18.

Authors

Eva Hulstaert^{1

2

3}, Anneleen Decock^{1

2}, Annelien Morlion^{1

2}, Celine Everaert^{1

2}, Kimberly Verniers^{1

2}, Justine Nuytens^{1

2}, Nele Nijs⁴, Gary P Schroth⁵, Scott Kuersten⁵, Stephen M Gross⁵, Pieter Mestdagh^{1

2

4}, Jo Vandesompele^{1

2

4}

Affiliations

¹ Center for Medical Genetics, Department of Biomolecular Medicine, OncoRNALab, Ghent University, C. Heymanslaan 10, 9000 Ghent, Belgium.
² Cancer Research Institute Ghent (CRIG), Ghent University, C. Heymanslaan 10, 9000 Ghent, Belgium.
³ Department of Dermatology, Ghent University Hospital, C. Heymanslaan 10, 9000 Ghent, Belgium.
⁴ Biogazelle, Technologiepark 82, 9052 Zwijnaarde, Belgium.
⁵ Illumina, San Diego, CA 92122, USA.

Abstract

Comprehensive transcriptome analysis of extracellular RNA (exRNA) purified from human biofluids is challenging because of the low RNA concentration and compromised RNA integrity. Here, we describe an optimized workflow to (1) isolate exRNA from different types of biofluids and (2) to prepare messenger RNA (mRNA)-enriched sequencing libraries using complementary hybridization probes. Importantly, the workflow includes 2 sets of synthetic spike-in RNA molecules as processing controls for RNA purification and sequencing library preparation and as an alternative data normalization strategy. For complete details on the use and execution of this protocol, please refer to Hulstaert et al. (2020).

Keywords: Molecular Biology; RNA-seq; Sequencing.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Extracellular Space / chemistry
Extracellular Space / genetics
Gene Expression Profiling / methods*
Gene Expression Profiling / standards
Humans
Polymerase Chain Reaction
RNA, Messenger / blood*
RNA, Messenger / isolation & purification
Reference Standards
Sequence Analysis, RNA* / methods
Sequence Analysis, RNA* / standards
Transcriptome / genetics*

Substances

RNA, Messenger