Depletion of tRNA-halves enables effective small RNA sequencing of low-input murine serum samples

Scientific Reports - Tập 6 Số 1
Alan Van Goethem1, Nurten Yigit1, Celine Everaert1, Myrthala Moreno‐Smith2, Liselot Mus1, Eveline Barbieri2, Frank Speleman1, Pieter Mestdagh3, Jason M. Shohet2, Tom Van Maerken3, Jo Vandesompele3
1Center for Medical Genetics Ghent (CMGG), Ghent University, Ghent, Belgium
2Department of Pediatrics, Section of Hematology-Oncology, Texas Children’s Cancer Center, Baylor College of Medicine, Houston, Texas, United States
3Cancer Research Institute Ghent (CRIG), Ghent University, Ghent, Belgium

Tóm tắt

AbstractThe 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.

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