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

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, R_amp , 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 R_amp 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 R_amp are useful, with the R_amp better evaluating mRNA integrity in this study.