RNA marker modifications reveal the necessity for rigorous preparation protocols to avoid artifacts in epitranscriptomic analysis

Abstract

Abstract The accurate definition of an epitranscriptome is endangered by artefacts resulting from RNA degradation after cell death, a ubiquitous yet little investigated process. By tracing RNA marker modifications through tissue preparation protocols, we identified a major blind spot from daily lab routine, that has massive impact on modification analysis in small RNAs. In particular, m6,6A and Am as co-varying rRNA marker modifications, appeared in small RNA fractions following rRNA degradation in vitro and in cellulo. Analysing mouse tissue at different time points post mortem, we tracked the progress of intracellular RNA degradation after cell death, and found it reflected in RNA modification patterns. Differences were dramatic between liver, where RNA degradation commenced immediately after death, and brain, yielding essentially undamaged RNA. RNA integrity correlated with low amounts of co-varying rRNA markers. Thus validated RNA preparations featured differentially modified tRNA populations whose information content allowed a distinction even among the related brain tissues cortex, cerebellum and hippocampus. Inversely, advanced cell death correlated with high rRNA marker content, and correspondingly little with the naïve state of living tissue. Therefore, unless RNA and tissue preparations are executed with utmost care, interpretation of modification patterns in tRNA and small RNA are prone to artefacts.

Autoren

Florian Richter
Johanna E Plehn
Larissa Bessler
Jasmin Hertler
Marko Jörg
Cansu Cirzi
Francesca Tuorto
Kristina Friedland
Mark Helm

DOI

10.1093/nar/gkab1150

eISSN

1362-4962

ISSN

0305-1048

Ausgabe der Veröffentlichung

8

Zeitschrift

Nucleic Acids Research

Sprache

en

Online publication date

2021

Paginierung

4201 - 4215

Datum der Veröffentlichung

2022

Status

Published

Herausgeber

Oxford University Press (OUP)

Herausgeber URL

http://dx.doi.org/10.1093/nar/gkab1150

Datum der Datenerfassung

2022

Titel

RNA marker modifications reveal the necessity for rigorous preparation protocols to avoid artifacts in epitranscriptomic analysis

Ausgabe der Zeitschrift

50

Datenquelle: Crossref

Abstract

The accurate definition of an epitranscriptome is endangered by artefacts resulting from RNA degradation after cell death, a ubiquitous yet little investigated process. By tracing RNA marker modifications through tissue preparation protocols, we identified a major blind spot from daily lab routine, that has massive impact on modification analysis in small RNAs. In particular, m6,6A and Am as co-varying rRNA marker modifications, appeared in small RNA fractions following rRNA degradation in vitro and in cellulo. Analysing mouse tissue at different time points post mortem, we tracked the progress of intracellular RNA degradation after cell death, and found it reflected in RNA modification patterns. Differences were dramatic between liver, where RNA degradation commenced immediately after death, and brain, yielding essentially undamaged RNA. RNA integrity correlated with low amounts of co-varying rRNA markers. Thus validated RNA preparations featured differentially modified tRNA populations whose information content allowed a distinction even among the related brain tissues cortex, cerebellum and hippocampus. Inversely, advanced cell death correlated with high rRNA marker content, and correspondingly little with the naïve state of living tissue. Therefore, unless RNA and tissue preparations are executed with utmost care, interpretation of modification patterns in tRNA and small RNA are prone to artefacts.

Addresses

Institute of Pharmaceutical and Biomedical Sciences, Johannes Gutenberg-University Mainz, Staudingerweg 5 55128 Mainz, Germany.

Autoren

Florian Richter
Johanna E Plehn
Larissa Bessler
Jasmin Hertler
Marko Jörg
Cansu Cirzi
Francesca Tuorto
Kristina Friedland
Mark Helm

DOI

10.1093/nar/gkab1150

eISSN

1362-4962

Externe Identifier

PubMed Identifier: 34850949
PubMed Central ID: PMC9071408

Funding acknowledgements

Deutsche Forschungsgemeinschaft: SPP1784
Deutsche Forschungsgemeinschaft: TRR319 RMaP
Deutsche Forschungsgemeinschaft: TP A06
JPND: Bmbf-Fkz 01ED1804
Deutsche Forschungsgemeinschaft: HE3397/17-1
Deutsche Forschungsgemeinschaft: TP C03

Open access

true

ISSN

0305-1048

Ausgabe der Veröffentlichung

8

Zeitschrift

Nucleic acids research

Schlüsselwörter

Animals
Mice
RNA
RNA, Ribosomal
RNA, Transfer
Artifacts
RNA Processing, Post-Transcriptional

Sprache

eng

Medium

Print

Open access status

Open Access

Paginierung

4201 - 4215

Datum der Veröffentlichung

2022

Status

Published

Publisher licence

CC BY

Datum der Datenerfassung

2021

Titel

RNA marker modifications reveal the necessity for rigorous preparation protocols to avoid artifacts in epitranscriptomic analysis.

Sub types

Research Support, Non-U.S. Gov't
research-article
Journal Article

Ausgabe der Zeitschrift

50

Files

https://academic.oup.com/nar/article-pdf/50/8/4201/43546907/gkab1150.pdf https://europepmc.org/articles/PMC9071408?pdf=render

Datenquelle: Europe PubMed Central

Abstract

The accurate definition of an epitranscriptome is endangered by artefacts resulting from RNA degradation after cell death, a ubiquitous yet little investigated process. By tracing RNA marker modifications through tissue preparation protocols, we identified a major blind spot from daily lab routine, that has massive impact on modification analysis in small RNAs. In particular, m6,6A and Am as co-varying rRNA marker modifications, appeared in small RNA fractions following rRNA degradation in vitro and in cellulo. Analysing mouse tissue at different time points post mortem, we tracked the progress of intracellular RNA degradation after cell death, and found it reflected in RNA modification patterns. Differences were dramatic between liver, where RNA degradation commenced immediately after death, and brain, yielding essentially undamaged RNA. RNA integrity correlated with low amounts of co-varying rRNA markers. Thus validated RNA preparations featured differentially modified tRNA populations whose information content allowed a distinction even among the related brain tissues cortex, cerebellum and hippocampus. Inversely, advanced cell death correlated with high rRNA marker content, and correspondingly little with the naïve state of living tissue. Therefore, unless RNA and tissue preparations are executed with utmost care, interpretation of modification patterns in tRNA and small RNA are prone to artefacts.

Date of acceptance

2021

Autoren

Florian Richter
Johanna E Plehn
Larissa Bessler
Jasmin Hertler
Marko Jörg
Cansu Cirzi
Francesca Tuorto
Kristina Friedland
Mark Helm

Autoren-URL

https://www.ncbi.nlm.nih.gov/pubmed/34850949

DOI

10.1093/nar/gkab1150

eISSN

1362-4962

Externe Identifier

PubMed Central ID: PMC9071408

Ausgabe der Veröffentlichung

8

Zeitschrift

Nucleic Acids Res

Schlüsselwörter

Animals
Artifacts
Mice
RNA
RNA Processing, Post-Transcriptional
RNA, Ribosomal
RNA, Transfer

Sprache

eng

Country

England

Paginierung

4201 - 4215

PII

6446534

Datum der Veröffentlichung

2022

Status

Published

Datum, an dem der Datensatz öffentlich gemacht wurde

2022

Titel

RNA marker modifications reveal the necessity for rigorous preparation protocols to avoid artifacts in epitranscriptomic analysis.

Sub types

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

Ausgabe der Zeitschrift

50

Datenquelle: PubMed

RNA marker modifications reveal the necessity for rigorous preparation protocols to avoid artifacts in epitranscriptomic analysis

Files

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