Differential ratio amplicons (Ramp) for the evaluation of RNA integrity extracted from complex environmental samples

Cholet, F., Ijaz, U. Z. and Smith, C. J. (2019) Differential ratio amplicons (Ramp) for the evaluation of RNA integrity extracted from complex environmental samples. Environmental Microbiology, 21(2), pp. 827-844. (doi:10.1111/1462-2920.14516) (PMID:30585386) (PMCID:PMC6392129)

Cholet, F., Ijaz, U. Z. and Smith, C. J. (2019) Differential ratio amplicons (Ramp) for the evaluation of RNA integrity extracted from complex environmental samples. Environmental Microbiology, 21(2), pp. 827-844. (doi:10.1111/1462-2920.14516) (PMID:30585386) (PMCID:PMC6392129)

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Abstract

Reliability and reproducibility of transcriptomics‐based studies are dependent on RNA integrity. In microbial ecology, microfluidics‐based techniques, such as the Ribosomal Integrity Number (RIN), targeting rRNA are currently the only approaches to evaluate RNA integrity. However, the relationship between rRNA and mRNA integrity is unknown. Here we present an integrity index, the Ratio Amplicon, Ramp, adapted from human clinical studies, to directly monitor mRNA integrity from complex environmental samples. We show, in a suite of experimental degradations of RNA extracted from sediment, that while the RIN generally reflected the degradation status of RNA the Ramp mapped mRNA degradation better. Furthermore, we examined the effect of degradation on transcript community structure by amplicon sequencing of 16S rRNA, amoA and glnA transcripts. We successfully sequenced transcripts for all three targets even from highly‐degraded RNA samples. While RNA degradation changed the community structure of the mRNA profiles, no changes were observed for the 16S rRNA transcript profiles. Since both RT‐Q‐PCR and sequencing results were obtained, even from highly degraded samples, we strongly recommend evaluating RNA integrity prior to downstream processing to ensure meaningful results. For this both the RIN and Ramp are useful, with the Ramp better evaluating mRNA integrity in this study.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Cholet, Fabien and Smith, Dr Cindy and Ijaz, Dr Umer Zeeshan
Authors: Cholet, F., Ijaz, U. Z., and Smith, C. J.
College/School:College of Science and Engineering
College of Science and Engineering > School of Engineering > Infrastructure and Environment
Journal Name:Environmental Microbiology
Publisher:Wiley
ISSN:1462-2912
ISSN (Online):1462-2920
Published Online:25 December 2018
Copyright Holders:Copyright © 2018 The Authors
First Published:First published in Environmental Microbiology 21(2): 827-844
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
652771Understanding microbial community through in situ environmental 'omic data synthesisUmer Zeeshan IjazNatural Environment Research Council (NERC)NE/L011956/1ENG - ENGINEERING INFRASTRUCTURE & ENVIR