Small ncRNA-Seq Results of Human Tissues: Variations Depending on Sample Integrity.

ABSTRACT

BACKGROUND: Although mature miRNAs are relatively stable in vivo, RNA degradation can have a substantial influence on small noncoding RNA (sncRNA) profiles. METHODS: Using different tissue storage conditions and RNA isolation procedures, we analyzed the integrity and quality of RNA isolates from human lung and heart tissues. We sequenced a total of 64 RNA samples and quantified the effect of RNA degradation, DNA contamination, and other confounding factors on the sncRNA-seq data. Besides microRNAs, other noncoding RNA species (piRNAs, tRNAs, snoRNAs, rRNAs) were investigated. RESULTS: Consistent with previous results, we found that the tissue specificity of microRNAs is generally well preserved. The distribution of microRNA isoforms was similar to the distribution of canonical forms. New miRNAs were more frequently discovered in degraded samples. sncRNA Reads generated from degraded samples mapped frequently to piRNAs, tRNAs, snoRNAs, or scaRNAs. Sequencing reads that were depleted of sncRNAs showed an increased mapping frequency to bacterial species. CONCLUSIONS: Our data emphasize the importance of sample integrity, especially for next-generation sequencing (NGS)-based high-throughput sncRNA profiles. For the prediction of novel miRNAs in particular, only samples with the highest RNA integrity should be used in order to avoid identification of false "miRNAs."