Guidelines for Successful Quantitative Gene Expression in Real- Time qPCR Assays

This chapter was developed to provide some important guidelines for studies with quantitative PCR (qPCR) using either dyes or probes, citing several essential components necessary for a good PCR assay. The efficiency and specificity of quantitative PCR (qPCR) depend on several parameters related to mRNA quantification that must be controlled to avoid mistakes in data interpretation. Avoiding contamination with proteins, carbohydrate and phenolic compounds during RNA extraction and purification processes will improve RNA quality and provide reliable results. Specific primers and sensible probes are also crucial to intensify efficiency, specificity and fluorescence. Other parameters such as the optimization of primer concentrations and efficiency primer curves must be done. During gene-expression profile quantification, qPCR assays using reference genes are required to normalize the target gene expression data. These reference genes are checked for stability to identify the most stable genes among a group of candidate genes that will be used to normalize the qPCR data, using programs such as geNorm, BestKeeper and NormFinder. Additionally, the choice of appropriate reference genes for a specific experimental condition is fundamental. The main aim of this chapter is to provide guidelines and highlight precautions to obtain a successful qPCR assays