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.

miRTrace reveals the organismal origins of microRNA sequencing data

“…Second, there have long existed protocols to separate the total pool of miRNAs from other transcripts [ 31 – 33 ], without the need for specific primers or probes. Third, miRNAs are very stable transcripts [ 34 ], detectable even in minute dried blood samples [ 35 ] or from samples several thousands of years old [ 36 ]. All these features combined make miRNAs very promising markers at the RNA level.…”

The future of Extracellular Vesicles as Theranostics – an ISEV meeting report

“…Deeper coverage will assist the analysis of rare sequences and give more confidence in the number of reads per transcript. Recently, it was reported that the classic ligation dependent library preparation method was biased to miRNAs [44], which calls for consideration of more recently developed methods such as capture and amplification by tailing and switching (CATS) [45,46]. During the data analysis phase, different algorithms/ calibration methods should be applied to characterize overlapping targets in order to improve the result confidence.…”

Untersuchung von miRNAs mithilfe getrockneter Blutstropfen

“…Eine Herausforderung für Hochdurchsatzsequenzierungen von miRNAs auf Basis von DBS war in der Vergangenheit sicherlich die sehr geringe Menge an kapillarem Blut und damit die geringe Menge an enthaltener RNA. Im Zuge der Entwicklung neuerer Protokolle gibt es jedoch erste Ansatzpunkte, die zukünftig die Hochdurchsatzsequenzierung von miRNAs auf Basis von DBS ermöglichen könnten [15].…”