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.
To address some of these issues, a kinetic approach to high-throughput relative quantification of gene expression has been successful. This chapter covers topics such as assay design and power analysis, RNA extraction and quality assessment, use of suitable internal controls, approaches to data analysis, and methods of ensuring comparable amplification efficiency across all samples, all of which are vital for successful qPCR. Most of these issues are not unique to the eye, and as such the aim of this chapter is to provide generalized advice for researchers using qPCR, as well as demonstrating how qPCR assays may be optimized for specific research challenges.
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