INTERACTION AND EFFECT OF ANNEALING TEMPERATURE ON PRIMERS USED IN DIFFERENTIAL DISPLAY RT-PCR

Differential display of mRNA is a simple, sensitive and powerful metho d to identify differentially expressed gene fragments. The main drawba ck of differential display is the lack of reproducibility and the-inab ility to read and compare complex gels, This issue results from employ ing unoptimized primer combinations and nonspecific amplification, mos t likely due to unavoidable low annealing temperatures. In order to di splay most of the expressed transcripts (80-120 band/lane), 26 differe nt 5' primers were used in conjunction with nine different 3' poly (dT ) primers. These primer combinations, used with the optimized annealin g temperature for each set of primers, produced highly reproducible ba nds. BSA has a direct effect on the number of bands resolved, Variatio ns in ramping time (9-40 s) had little or no effect on the resolution and reproducibility of differential display.