Small ncRNAs (sncRNAs) offer great hope as biomarkers of disease and response to treatment. This has been highlighted in the context of several medical conditions such as cancer, liver disease, cardiovascular disease, and central nervous system disorders, among many others.
Researchers from McGill University assessed several steps involved in the development of an ncRNA biomarker discovery pipeline, ranging from sample preparation to bioinformatic processing of small RNA sequencing data. At each stage, they evaluated the pros and cons of different techniques that may be suitable for different experimental designs. Evaluation methods included quality of data output in relation to hands-on laboratory time, cost, and efficiency of processing.
Illustration of study design and samples. Human biological samples (N = 45) were included in the present study. a Peripheral blood from a single individual was split into 11 aliquots (technical replicates) to test three different small RNA library purification methods: Novex TBE PAGE gel (N = 3), Pippin Prep automated gel system (PPS) (N = 4), and AMPure XP beads ((N = 3). Sample C1 (control-human brain) (N = 1), sample AC (control-no purification method) (N = 1). b Peripheral blood from a single individual was split into 5 aliquots (technical replicates) to test optimal amounts of RNA input: (1 μg), (0.5 μg), (0.25 μg), (0.1 μg), and (0.05 μg). All libraries were purified using the PPS system. c Peripheral blood samples from 15 healthy volunteers (biological replicates) to test the effects of RNA integrity. Samples were split into 5 groups (N = 3) with average RIN values of 9, 7, 5.4, 2.2 and 0. All libraries were purified using AMPure XP beads. d Peripheral blood samples from 12 healthy volunteers (biological replicates) to test effects of sequencing coverage. Samples sequenced on both a HiSeq2500 (N = 12) and MiSeq (N = 12) Illumina sequencers. All libraries were purified using AMPure XP beads. e Human whole-blood (N = 4), brain (N = 4), heart (N = 4) and liver (N = 4) tissues to test expression and tissue specificity of small ncRNAs. All libraries were purified using AMPure XP beads.
The 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.
This study provides tools and outcome metrics that will aid researchers and clinicians in choosing an appropriate and effective high-throughput sequencing quantification method for various study designs, and overall generating valuable information that can contribute to our understanding of small ncRNAs as potential biomarkers and mediators of biological functions and disease.