miRNome analysis using real-time PCR.

MicroRNAs (miRNAs) are short RNA molecules that regulate gene expression in eukaryotic organisms, thus influencing physiological mechanisms such as development, cell proliferation, cell death, and cell differentiation. The importance of the gene regulatory system operated by miRNAs is emerging as a central topic in the setting of several diseases included infectious disease and cancer. The different techniques used for the study of the entire "miRNome" give the opportunity to go better inside these novel mechanisms of gene expression regulation. In the following method we describe a protocol based on quantitative real-time PCR (qRT-PCR) with SYBR(®) green technology, to specifically analyze the expression levels of only those miRNAs that target genes involved in CTLs biogenesis and functions. Through an in silico approach, we designed a custom microRNA qPCR panel focused on those miRNAs relevant in regulation of CTLs-specific pathways. The panel we created was customized by EXIQON, since this company proposed a method based on the use of LNA enhanced primers, which guarantee increased affinity and specificity for each microRNA. The advantage of this protocol with respect to a whole miRNome analysis consists in the possibility to evidence weaker signals that otherwise would be secreted and remove the noise itself generated by other miRNAs not directly involved in the regulation of CTLs-specific pathways. This panel can be applicable in the study of CTLs behavior in pathological conditions such as infectious disease and cancer or can be used to characterize changes in patients' immune responsiveness after therapeutic intervention in order to understand the molecular mechanisms underlying these effects.