Optimizing primer--probe design for fluorescent PCR.

TaqMan, a variation of fluorescent PCR, is a powerful tool for gene expression and polymorphism studies. Here we describe the design and evaluation of 27 new TaqMan primer-probe sets for rat genes that play a key role in neural signaling. These newly designed and synthesized probes were tested and then used for quantification of RNA isolated from rat brain. The usual length of common TaqMan probes is 25 bases or less. In these studies we constructed probes with lengths of 25-39 bases to span exon-exon junctions of nucleic acids to avoid the influence of DNA contamination upon the RNA quantification. The specific sequences at these positions required probes of these lengths to optimize hybridization. We found that the relocation of the quencher from the traditional 3' position to an internal one increases the sensitivity of probe up to 30 fold. Substitution of 6-carboxyfluorescein with Alexa Fluor 488 as fluorophore and TAMRA with non-fluorescent quencher dabcyl was also investigated. We also describe the evaluation of part of a newly designed set of 27 TaqMan primer-probes for the measurement of differences in gene expression levels in samples from the caudate putamen region of rat brain after 'binge' paradigm cocaine administration. Cocaine-induced alterations in expression of c-fos and preprodynorphin mRNAs measured by TaqMan were confirmed by ribonuclease protection assay.