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

Abstract

26Background 27 Reliability and reproducibility of transcriptomics-based studies are highly dependent on the 28 integrity of RNA. Microfluidics-based techniques based on ribosomal RNA such as the RNA 29 Integrity Number (RIN) are currently the only approaches to evaluate RNA integrity. 30 However, it is not known if ribosomal RNA reflects the integrity of the meaningful part of the 31 sample, the mRNA. Here we test this assumption and present a new integrity index, the Ratio 32 amplicon, R amp , to monitor mRNA integrity based on the differential amplification of long to 33 short RT-Q-PCR amplicons of the glutamine synthetase A (glnA) transcript. 34Results 35 We successfully designed and tested two R amp indexes targeting glnA transcripts. We showed 36 in a suite of experimental degradations of RNA extracted from sediment that while the RIN in 37 general did reflect the degradation status of the RNA well the R amp mapped mRNA 38 degradation better as reflected by changes in Reverse Transcriptase Quantitative PCR (RT-Q-39 PCR) results. Furthermore, we examined the effect of degradation on transcript community 40 structure by amplicon sequencing of the 16S rRNA, amoA and glnA transcript which was 41 successful even form the highly-degraded samples. While RNA degradation changed the 42 community structure of the mRNA profiles, no changes were observed between successively 43 degraded 16S rRNA transcripts profiles. 44 Conclusion 45As demonstrated, transcripts can be quantified and sequenced even from highly degraded 46 samples. Therefore, we strongly recommend that a quality check of RNA is conducted to 47 ensure validity of results. For this both the RIN and R amp are useful, with the R amp better 48 evaluating mRNA integrity in this study. 49 50 3 51 52Background 53 A key question in environmental microbiology is to determine the functioning and activity of 54 microbial communities. While genomic approaches have resulted in an unprecedented 55 understanding of their structure and complexity [1], they do not inform of the actual activity 56 and functioning at a given time. In this case targeting the transcriptome, that is the subset of 57 genes that are actively transcribed at a given time, is more informative. While there can be 58 substantial post translational regulation that may prevent final protein synthesis and/or 59 activity, gene expression is the direct link between the genome and the function it encodes 60 and therefore is a stronger link to activity than DNA approaches alone [2]. As a result 61 transcriptomics based approaches are widely used to assess microbial activity and functioning 62 in the environment [3, 4]. The premise is that that messenger RNA (mRNA) turn-over within 63 cells is rapid, ranging from a few minutes to less than an hour [5]. As such a snap-shot of the 64 transcriptome reflects the cells transcriptional response to its surrounding environment and 65 metabolic needs at a given time. 66A challenge for all transcript-based studies, not least for those from environmental...