Analysis of miRNA Expression Using Digital PCR and the QuantStudio™ 3D Digital PCR System

miRNAs are short, single-stranded RNA molecules that function as posttranscriptional regulators of gene expression. miRNAs represent ideal biomarkers since they can also circulate in the bloodstream as well as in other biological fluids such as urine, saliva, and cerebrospinal fluid.miRNAs play an important role in the regulation of immune cells including cytotoxic T-lymphocytes. Circulating miRNAs can be analyzed by Real-Time PCR or microarray profiling; however data normalization represents still an unsolved problem due to the lack of widely validated house-keeping miRNAs candidates.Digital PCR (dPCR) is an end-point PCR method that is used for absolute quantification. In this chapter we will describe the applications of Digital PCR for the analysis of miRNAs that can influence immune response in serum samples and we will report a specific protocol that can be used to analyze miRNAs using the QuantStudio™ 3D Digital PCR System. The advantage of this method consists in the possibility to highlight weaker differences in miRNA circulating molecules that can be useful to monitor CTLs behavior in pathological conditions or after therapeutic intervention.