Next Generation Sequencing Analysis of Total Small Noncoding RNAs from Low Input RNA from Dried Blood Sampling.

Abstract

Circulating miRNAs are favored for biomarker candidates as they can reflect tissue specific miRNA dysregulation in disease contexts. Moreover, they have the additional advantage that they can be monitored in a minimally invasive manner. Blood-borne miRNAs are therefore currently characterized to identify, describe, and validate their potential suitability as biomarkers; however, sampling and as well miRNA detection methods limit these studies in terms of sensitivity but also practicability in clinical, at-home, or low-resource sampling of high-quality circulating RNA samples. We describe here a novel and innovative method of circulating RNA microsampling from minimal volume dried blood samples with direct enrichment for small RNA fractions in combination with ligation free library preparation. We evaluated crucial parameters for efficient library preparation from low RNA inputs of 50 pg for efficient dissection not only of miRNAs but also isomiRs, piRNAs, and lincRNAs. We compared these data to classical microarrays and characterize the technical reproducibility and its sensitivity. We demonstrate and evaluate a method for easy low resource sampling and NGS analysis of circulating RNAs providing a powerful tool for massive cohort and remote patient monitoring.