ABSTRACT
This chapter addresses a range of problems that arise with quantifying gene expression with quantitative PCR (qPCR), drawing upon the author’s experience using the eye as a model system. The eye (or more specifically the light-sensitive retina), as well as mediating the primary events of vision, provides an accessible model of the nervous system. However, the lightsensitive nature of this tissue makes analysis of ocular tissues difficult as dynamic changes in gene expression may occur during tissue collection. The diverse cellular makeup of the retina also presents a problem in the form of assessing gene expression in a heterogeneous tissue. When dealing with models of retinal degeneration, these problems are compounded by very small tissue samples and, furthermore, when the pathways of interest involve numerous transcripts, high-throughput assays become essential.
