In a highly accessed paper in BMC Medical Genomics, scientists from McGill University and EMBL tested several steps to find the most robust pipeline for discovering small non-coding RNAs (sncRNAs) that might be useful as biomarkers. As part of this effort, they evaluated several sizing options for microRNAs.
“Biomarker discovery: quantification of microRNAs and other small non-coding RNAs using next generation sequencing” comes from lead author Juan Pablo Lopez, senior author Carl Ernst, and several collaborators. The team sequenced 45 samples with Illumina platforms and validated the sequence data with qRT-PCR. “Our results show that good quality sequencing libraries can be prepared from small amounts of total RNA and that varying degradation levels in the samples do not have a significant effect on the overall quantification of sncRNAs via NGS,” the authors report.
Size selection of small RNAs has long been a challenge in workflows like these, as microRNAs and other sncRNAs tend to be very close in size to the adapters and other artifacts that must be removed to get the best results. In this study, scientists compared the Pippin Prep from Sage Science to Novex TBE PAGE gels and AMPure XP beads. Noting that their goal was to evaluate pros and cons of each technique, rather than choosing the best, they report, “We were able to obtain good quality sequencing libraries for all samples, but nonetheless, we found significant differences across purification methods.”
One of those differences was library yield. “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 team writes.
After sequencing, the team assessed reads produced from each sizing method, as well as from a control library with no purification step. “Libraries prepared using [Pippin] gave the highest number of total reads with an average of 11.8 M reads per sample, while with the others we obtained only an average of 8.8 M (Novex), 9.1 M (AMPure) and 8.5 M (no purification),” Lopez et al. write. They add that Pippin sizing also identified more distinct miRNAs than any other protocol, and had the highest specificity to miRNAs.
Based on that, the team recommends Pippin Prep for medium-size projects. Our PippinHT was released after this project was completed and is a good option for scientists interested in the same high-quality, automated approach with significantly higher throughput.