Strategies to normalize miRNA expression in serum and plasma

MicroRNA(miRNA) are short non-coding single stranded RNA molecules that regulate gene expression at the post transcriptional level.  They are known to play a critical role in multiple biological processes including proliferation, differentiation, apoptosis and hematopoiesis. Dysregulation of miRNA expression therefore is associated with a number of pathological conditions such as inflammation, cardiovascular diseases, neurological disorders and several types of cancers. miRNAs are also found to be extremely stable in body fluids such as serum, plasma, urine, breast milk and saliva. Extracellular miRNAs are either packed into exosomes or loaded into high-density lipoproteins, or bound by AGO2 proteins outside on vesicles. These modes of action protect miRNAs from degradation and improve their stability. Because of their stability in body fluids, miRNAs are gaining importance as biomarkers in liquid biopsies. Real time qPCR technologies reliably and accurately detect and measure miRNAs levels from body fluids.  However, since no single miRNA is known to be constitutively expressed in body fluids, normalizing real time qPCR data for comparing miRNA expression levels in different samples continues to be a challenge. In this study, we demonstrate a systematic methodology to identify and verify stably expressed miRNAs in body fluids.  The stably expressed miRNAs are then used to normalize miRNA expression profiles in plasma.  We illustrate this workflow for identifying miRNA biomarkers in plasma using the TaqMan Human Advanced miRNA Panels on 96-well plates, Taqman Array Cards (TAC), and Open Array plates.  These different formats provide throughput flexibility for miRNA biomarker discovery research programs.

Learning Objectives:

How to identify stably expressed/endogenous miRNAs in serum and plasma

How to use spike-in controls to normalize miRNA expression in difficult samples.

Workflow to identify normalizers in serum and plasma using TaqMan® Array Cards, Plates and OpenArray™