Size selection is a critical step in NGS pipelines, but may be most challenging for studies of small RNAs. The concept behind size selection is simple: separate a sheared DNA or cDNA sample by fragment size, and then use the resulting sizes to remove unwanted fragments. This is a tried-and-true way to get rid of the unwanted adapter-dimer content and other artifacts that would otherwise contaminate your NGS libraries and waste sequencing effort. But what do you do when the fragments you’re trying to study are almost the same size as that adapter-dimer content?
After going undetected for decades, small RNAs are now understood to be incredibly important biological targets. From microRNAs to small nuclear RNAs and more, these diminutive molecules pack a punch, often in regulatory functions. But the pipelines set up to study DNA fragments hundreds to thousands of bases in size don’t always translate to these much smaller snippets of RNA.
Many labs still rely on manual gels for DNA sizing, but these typically don’t offer the resolution needed to distinguish between, say, a band of miRNAs and one of adapter-dimers. Automated size selection methods have been more promising, though they don’t all produce the same results.
A recent paper in BMC Medical Genomics reports a study comparing size selection methods for miRNA and small non-coding RNA discovery. The scientists, from McGill University and the European Molecular Biology Laboratory, compared the Pippin Prep from Sage Science to Novex TBE PAGE gels and AMPure XP beads. They found significant differences in library yield between the systems: “The four libraries purified using [Pippin] also showed single peaks corresponding to miRNAs, but these libraries contained more than 50 times more product after purification, as compared to the Novex gel method,” the authors wrote. They noted that Pippin sizing led to higher miRNA specificity as well as the identification of more distinct miRNAs than the other methods.
Check out these papers using automated size selection for small RNAs to isolate miRNAs:
- KRAS-dependent sorting of miRNA to exosomes
- MicroRNAs in the oriental fruit fly, Bactrocera dorsalis: extending Drosophilid miRNA conservation to the Tephritidae
- FOXO1 regulates expression of a microRNA cluster on X chromosome