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

• Published in: STAR protocols Vol. 2; no. 2; p. 100475
• Main Authors: Hulstaert, Eva, Decock, Anneleen, Morlion, Annelien, Everaert, Celine, Verniers, Kimberly, Nuytens, Justine, Nijs, Nele, Schroth, Gary P., Kuersten, Scott, Gross, Stephen M., Mestdagh, Pieter, Vandesompele, Jo
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 18.06.2021; Elsevier
• Subjects: Extracellular Space - chemistry; Extracellular Space - genetics; Gene Expression Profiling - methods; Gene Expression Profiling - standards; Humans; Molecular Biology; Polymerase Chain Reaction; Protocol; Reference Standards; RNA, Messenger - blood; RNA, Messenger - isolation & purification; RNA-seq; Sequence Analysis, RNA - methods; Sequence Analysis, RNA - standards; Sequencing; Transcriptome - genetics; RNA-seq; Molecular Biology; Sequencing
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2021.100475

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). [Display omitted] •Extracellular RNA from biofluids has a low concentration and a compromised integrity•An optimized workflow for mRNA capture sequencing of human biofluids is provided•Synthetic spike-in RNA molecules serve as processing controls•Spike-in RNAs allow for data normalization and calculation of mRNA concentration 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.