Real-Time PCR Assay for the Analysis of Alternative Splicing of Immune Mediators in Cancer.

Alternative splicing evolved as a very efficient way to generate proteome diversity and to regulate cell homeostasis from a limited number of genes. Moreover, changes in the relative amounts of different splice variants derived from the same pre-mRNA are a hallmark in cancer, and aberrant expression of alternatively spliced mRNAs has been linked to cancer initiation and progression. Therefore, splice variants are critical tools to assess disease progression and clinical prognosis, and hold great promise as potential targets for therapeutic intervention. In order to understand the role that such splice variants play in cancer, it is vital to be able to accurately quantify their expression levels in different cell types and organs, both in normal conditions and in disease. In this chapter we describe a protocol to efficiently detect, analyze, and quantify alternative splicing patterns of immune mediators such as chemokines, cytokine and their receptors and ligands in cancer by quantitative PCR.