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

Abstract

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.

Identification of sexually dimorphic gene expression in brain tissue of the fish Leporinus macrocephalus through mRNA differential display and real time PCR analyses

“…Although DD also has some disadvantages, as the possibility of isolation ''false-positive'' transcripts (PCR products that appear to be differentially expressed on the gel, but that cannot be verified when subsequent expression analyses are performed) (Liang 1998, Pardee andMcClelland 1999), technical improvements have been increasing its successful application. Modifications which have been proposed to improve differential display include the use of primers tailored to amplify members of a particular gene family (Jurecic 1996), the optimization of annealing temperature (Malhotra et al 1998), and optimization of primer design (Graf et al 1997).…”

Multidimensional Differential Display via Ion-Pair Reversed-Phase Denaturing High-Performance Liquid Chromatography

“…The method was first introduced by Liang and Pardee (1) and has subsequently been modified and improved (11,17). The main drawbacks of this method are the lack of reproducibility (18), the inability to read and compare complex gels (18), and difficulties in recovering the desired bands from gels. Optimized primers and annealing temperatures can reduce the number of false positives (18) and we believe that using IP RP DHPLC will overcome the problems related to comparing and recovering the bands from complex gels.…”

Innate immunity: Eggs of Manduca sexta are able to respond to parasitism by Trichogramma evanescens

“…Each of the cDNA types was used as template for eight different PCR reactions. Each PCR reaction was primed with one of eight forward arbitrary primers, consisting of a random decamer tailed at its 5 0 end with an arbitrary primer sequence as described by Bauer et al (1993) and Malhotra et al (1998) (Table S1), plus dVA, dVT, dVC, or dVG primers (where V may be A, G, or C) as the degenerate primer. As template we used 2 mL of the synthesized 1st strand cDNA.…”