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Abstract
The ongoing ascent of sequencing technologies has enabled researchers to gain unprecedented insights into the RNA content of biological samples. MiRNAs, a class of small non-coding RNAs, play a pivotal role in regulating gene expression. The discovery that miRNAs are stably present in circulation has spiked interest in their potential use as minimally-invasive biomarkers. However, sequencing of blood-derived samples (serum, plasma) is challenging due to the often low RNA concentration, poor RNA quality and the presence of highly abundant RNAs that dominate sequencing libraries. In murine serum for example, the high abundance of tRNA-derived small RNAs called 5′ tRNA halves hampers the detection of other small RNAs, like miRNAs. We therefore evaluated two complementary approaches for targeted depletion of 5′ tRNA halves in murine serum samples. Using a protocol based on biotinylated DNA probes and streptavidin coated magnetic beads we were able to selectively deplete 95% of the targeted 5′ tRNA half molecules. This allowed an unbiased enrichment of the miRNA fraction resulting in a 6-fold increase of mapped miRNA reads and 60% more unique miRNAs detected. Moreover, when comparing miRNA levels in tumor-carrying versus tumor-free mice, we observed a three-fold increase in differentially expressed miRNAs.
Highlights
The ongoing ascent of sequencing technologies has enabled researchers to gain unprecedented insights into the RNA content of biological samples
Libraries prepared from serum samples that potently hinders the detection of other small RNA species, targeted depletion of these 5′tRNA halves could prove highly efficient
In this study we optimized and compared two protocols for the selective depletion of 5′tRNA halves in RNA isolated from murine serum samples of which only one protocol was successful in targeted depletion of 5′tRNAs halves
Summary
In RNA samples depleted using RNase H, 14.6% of mapped reads were assigned to 5′tRNA halves, only 0.9% to miRNA and 84.0% to other small RNA fragments (Fig. 4a). For RNA samples depleted using beads, we observed that 23% of mapped reads map to 5′tRNAs halves, 35% to miRNAs and 42% map to other small RNA fragments (Fig. 4a). These results are illustrated in a frequency histogram of the RNA length distribution. Depletion using RNase H resulted in a strong reduction in both miRNA reads and detected miRNAs as compared to the control samples, with a total of 40 detected miRNAs in depleted samples (Table 1). Only in depleted samples, sensitivity was high enough to detect differentially expressed human-specific miRNAs like hsa-miR-144-5p, a miRNA that is reported to be overexpressed in human neuroblastoma cells[24]
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