Identification of Stable Reference Genes for Toxicogenomic and Gene Expression Analysis.

Gene expression analysis has been becoming a popular method for studying gene function and response to different environmental stresses, including toxin/pollution exposure. Selection of a suitable reference gene is critically important for gene expression analysis due to that wrong reference genes will cause misleading and even wrong conclusion. A good reference gene should be a more stable reference gene, particularly during the toxicant exposure treatment and/or other investigation condition. In this chapter, a step-by-step protocol is present for primer design, reverse transcription PCR, primer efficiency and specificity test, qRT-PCR, and the strategy for identifying most stable reference genes for toxicogenomic and gene expression analysis. The detailed method for determining the primer gene specificity and primer efficiency are also presented in this chapter. Low primer efficiency will affect the fold changes during gene expression analysis; however, it does not affect the conclusion, up- or downregulation. Choosing a wrong reference gene may result in wrong conclusion.