Differential Display

The technique of differential display is designed to determine the complement of genes being expressed (mRNAs) by a tissue or an organ at a given point in time. The establishment of differential display is dependent on the random amplification and subsequent size separation of cDNA molecules (Liang et al. 1992). With RT-PCR amplification with specific oligo-T primers (one- or two-base anchored oligo-dT primers, such as oligo-T-XC, oligo-T-XG, oligo-T-XT, and oligo-T-XA; X = G/A/C), four separate cDNA synthesis reactions are performed. These cDNA synthesis reactions form the four pools of cDNA for one original mRNA population. The resulting cDNA molecules from each RT-PCR reaction are amplified, using the same primer of the reverse transcription step plus randomly chosen primers. Because the randomly chosen primers will anneal at various locations upstream of the oligo-T site, many individual cDNA fragments of different sizes are amplified in each PCR reaction. cDNA fragments derived from different original mRNA populations are sized and then separated on parallel gels to analyze the presence of unique bands. The differentially expressed cDNA fragments can be excised from gels, cloned, and further characterized by a variety of technologies based on different purposes, such as in situ hybridization, sequences, and Northern blot analysis.

Differential display is a useful tool in identifying region-specific mRNA transcripts in brain. The molecular markers for these regions can be found by screening for gene expression in specific brain regions or nuclei (Mizushima et al. 2000; Tochitani et al. 2001). In addition, under different stimulation or behavioral conditions, the changes in gene expression can be explored by differential display (Hong et al. 2002; Q. R. Liu et al. 2002; Mello et al. 1997; Tsai et al. 2002). This technique has even been adapted to indicate the RNA expression profile of an individual neuron (Eberwine et al. 1992). Many genes related to ischemia or Alzheimer's disease in the CNS were also isolated by means of differential display (Doyu et al. 2001; Imaizumi et al. 1997; Tanaka et al. 2002). Genes that are activated in response to chronic drug treatment (e.g., opiates or antidepressants) can also be identified this way. Furthermore, as described later in this chapter (see "Microarray Technology"), streamlined technologies (gene arrays) are now available for this purpose.

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