Generating Robust Results From qPCR Analysis Of MicroRNAs In Biofluids

Ditte Andreasen, Thorarinn Blondal, Maria Wrang Theilum, Niels Tolstrup, Jörg Krummheur, Nana Jacobsen, Peter Mouritzen Exiqon A/S, Denmark

Abstract

microRNAs (miRNAs) constitute a class of small cellular RNAs (typically 19-23 nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the cellular transcriptome is regulated by miRNAs, although they are relatively few in number (less than 2000 human miRNAs).
The high relative stability of miRNAs in common clinical source materials (FFPE blocks, plasma, serum, urine, saliva, etc.) and the ability of microRNA expression profiles to accurately classify discrete tissue types and specific disease states have positioned microRNAs as promising new biomarkers for diagnostic application. Furthermore miRNAs have been shown to be actively exported from tissues into the circulation with the development of pathology, through a variety of mechanisms including exosome and microvesicle transport, and complexing with RNA binding proteins or HDL.
The main challenge in detecting miRNA is their small size, no longer than one standard PCR primer. Furthermore, many miRNAs belong to families with only a few mismatches between members, and the G/C content varies greatly. To overcome these obstacles and facilitate discovery and clinical development of miRNA-based biomarkers in biofluids, we developed an LNA™-based miRNA PCR platform with unparalleled sensitivity and robustness. The platform uses a single universal RT reaction per sample combined with two target specific, LNA™ enhanced PCR primers per miRNA assay to profile human miRNAs. This facilitates high-throughput profiling of miRNAs from important clinical sources, with high specificity and sensitivity, without the need for pre-amplification. We have recently further developed the technology with a new RNA extraction kit and improved qPCR reagents, further increasing sensitivity and specificity while allowing high-throughput processing with the use of ambient temperature stacking systems.
Using the LNA™ PCR system, we have profiled thousands of biofluid samples. An extensive QC system has been implemented in order to secure technical excellence and reveal any unwanted bias in the dataset. We will present our approaches to sample handling, qPCR technology, data normalization and studies of pre-analytical variables such as hemolysis.