Differential amplicons for the evaluation of RNA integrity extracted from complex environmental samples

Abstract

BackgroundReliability and reproducibility of transcriptomics-based studies are highly dependent on the integrity of RNA. Microfluidics-based techniques based on ribosomal RNA such as the RNA Integrity Number (RIN) are currently the only approaches to evaluate RNA integrity. However, it is not known if ribosomal RNA reflects the integrity of the meaningful part of the sample, the mRNA. Here we test this assumption and present a new integrity index, the Ratio amplicon, Ramp, to monitor mRNA integrity based on the differential amplification of long to short RT-Q-PCR amplicons of the glutamine synthetase A (glnA) transcript.\n\nResultsWe successfully designed and tested two Ramp indexes targeting glnA transcripts. We showed in a suite of experimental degradations of RNA extracted from sediment that while the RIN in general did reflect the degradation status of the RNA well the Ramp mapped mRNA degradation better as reflected by changes in Reverse Transcriptase Quantitative PCR (RT-Q-PCR) results. Furthermore, we examined the effect of degradation on transcript community structure by amplicon sequencing of the 16S rRNA, amoA and glnA transcript which was successful even form the highly-degraded samples. While RNA degradation changed the community structure of the mRNA profiles, no changes were observed between successively degraded 16S rRNA transcripts profiles.\n\nConclusionAs demonstrated, transcripts can be quantified and sequenced even from highly degraded samples. Therefore, we strongly recommend that a quality check of RNA is conducted to ensure validity of results. For this both the RIN and Ramp are useful, with the Ramp better evaluating mRNA integrity in this study.