Analysis of gene expression in cultured primary neurons

Abstract

This chapter highlights the use of the reverse transcription-polymerase chain reaction (RT-PCR) assay in analyzing the effects of cytokines on neuronal gene expression. Recent progress in molecular biologic techniques has enhanced the utility of primary cultures. Amplification of signals by PCR after RT of RNA to cDNA provides a way to obtain more information than previous methods, and allows the use of small numbers of cultured cells. Furthermore, the RT-PCR may be used to simultaneously determine changes in the expression of multiple neuronal genes. Thus, this approach circumvents some of the limitations of primary culture and greatly enhances the ability to study neuronal gene expression after experimental manipulation. Although the assay introduced in the chapter is only semiquantitative, quantitative results are easily obtained using competitive PCR or Southern analysis after PCR, and making the adjustment of RNA in each sample by calculating ratio of each gene signal to an internal control (such as p-actin). Different ways of preparing neuronal cultures have been introduced and various neuronal stem cells, which are capable of self-replication and generate committed cells, are becoming available.