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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.

Microarrays play an important role in the study of the transcriptome in a variety of conditions and species. This is documented by their widespread use over the last decade in areas such as cancer, immunology, neurological disorders, renal diseases, and many others, directed towards the new frontiers of personalized medicine and drug discovery. The following method covers a specific application of microarrays in the field of immunology, focusing on the study of antigen-induced T cell differentiation in response to viral or bacterial infection, and in the context of cancer. This protocol allows, through an "Omics" strategy, the study of the transcriptome of CTLs, concentrating only on the expression profiles of those genes more likely to be involved in CTL action. Since the biological question, in this case, is very specific, the advantage of this protocol with respect to a more traditional whole transcriptome microarray experiment is to remove the noise coming from all the genes not directly involved in the CTLs-specific pathways, highlighting weaker signals that otherwise would be hidden by the noise itself. To address this issue we have accurately selected all the CTLs-specific pathways, extracted all the genes belonging to them, and designed a CTL-specific microarray, based on all known validated transcripts deriving from these genes. This microarray has been built for the Agilent Technologies microarray platform, the only one that, to our knowledge, at present allows autonomously designing a completely customizable microarray. We used it in the context of renal cell carcinoma (RCC), but surely it will find several more applications in many other cancers and in the context of viral and bacterial infection.