Methods for quantifying gene expression in ecoimmunology: From qPCR to RNA-Seq

Synopsis Historically, the use of cutting-edge molecular techniques to study immunological gene expression and related cellular pathways has been largely limited to model organisms. Few studies have been performed that quantify the molecular immunological responses of non-model species, especially in response to environmental factors, life-history events, or exposure to parasites. This dearth of information has largely occurred due to the lack of available non-model species-specific gene sequences and immunological reagents and also due to prohibitively expensive technology. However, with the rapid development of various sequencing and transcriptomic technologies, profiling the gene expression of nonmodel organisms has become possible. Technologies and concepts explored here include an overview of current technologies for quantifying gene expression, including: qPCR, multiplex branched DNA assays, microarrays, and profiling gene expression (RNA sequencing [RNA-Seq]) based on next-generation sequencing. Examples of the advancement of these technologies in non-model systems are discussed. Additionally, applications, limitations, and feasibility of the use of these methodologies in non-model systems to address questions in ecological immunology and disease-ecology are specifically addressed.