RT-PCR Analyses of Differential Gene Expression in ES-Derived Neural Stem Cells

Reverse transcription and the polymerase chain reaction (RT-PCR) provides a very sensitive method to identify known genes that are both upregulated and downregulated during neuronal differentiation. First strand cDNAs are generated in the initial reaction with reverse transcriptase in the presence oligo-dT primed RNAs. The cDNA provides the template for amplification of gene specific targets with the appropriate primers. The purity of the RNA is extremely important to the success of these procedures. The presence of chromosomal DNA in the RNA preparation will influence the reliability of both RNA concentration and the PCR products generated from these RNA templates in the reverse transcription reactions. Many parameters in PCR have to be optimized for each gene specific primer pair and template; they include primer and MgCl₂ concentrations, primer annealing temperatures, and the number of PCR cycles (1 ,2 ).