Palatal Dysmorphogenesis: Quantitative RT-PCR

The formation of the secondary palate requires coordinated expression of genes involved in processes such as cell proliferation, differentiation, and production of extracellular matrix proteins. In order to study the mechanisms through which teratogenic agents induce cleft palate, the regulation and expression of these genes needs to be examined and compared between control and treated embryos. The expression of specific mRNAs can be examined by reverse transcription polymerase-chain reaction (RT-PCR) and this chapter presents an application of RT-PCR for quantitating the level of mRNA. The sensitivity of this method allows the investigator to evaluate gene expression in individual embryos using dissected tissues from the specific regions/ anatomic structures affected by the teratogen. Even extremely small specimens can usually be used to quantitate mRNAs for several genes. Other methods, such as the Southern blot, generally require pooling of multiple embryos and may not provide information for the isolated, specific target tissue. The quantitative method described in this chapter is derived from the method published by Vanden Heuvel (1). The modifications we introduce improved the performance in our laboratory and may enhance the reliability, specificity, and sensitivity of this approach. This introduction provides an overview of the issues of experimental design that are important to the success of the method, including internal standard design, primer selection, image acquisition and analysis. A basic introduction to the RT-PCR method can be found in one of the many resource and protocol books available on this topic, including a volume edited by McPherson et al. (2).