New index for the evaluation of mRNA integrity

Background Reliability and reproducibility of transcriptomics-based studies are highly dependent on the integrity of RNA. Microfluidics-based techniques, such as RNA Integrity Number (RIN) based on ribosomal RNA 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 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. ResultsWe successfully designed and tested two Ramp indexes targeting glnA transcripts. We showed in a range of experimental degradations of sediment RNA that the Ramp successfully monitored RNA integrity and overall correlated better with quantitative changes in 16S rRNA and amoA transcripts than the RIN in experimentally degraded environmental RNA. However, the RIN 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. Amplicon sequencing of 16S rRNA, amoA and glnA transcript was successful even for 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.ConclusionAs 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.