Interaction and effect of annealing temperature on primers used in differential display RT-PCR.

Differential display of mRNA is a simple, sensitive and powerful method to identify differentially expressed gene fragments. The main drawback of differential display is the lack of reproducibility and the inability to read and compare complex gels. This issue results from employing unoptimized primer combinations and non-specific amplification, most likely due to unavoidable low annealing temperatures. In order to display most of the expressed transcripts (80-120 bands/lane), 26 different 5' primers were used in conjunction with nine different 3' poly (dT) primers. These primer combinations, used with the optimized annealing temperature for each set of primers, produced highly reproducible bands. BSA has a direct effect on the number of bands resolved. Variations in ramping time (9-40 s) had little or no effect on the resolution and reproducibility of differential display.