Quantitative real-time PCR assay of SNAP-25 isoform transcripts. Quantitative RT-PCR demonstrated specific and equivalent amplification of SNAP-25 isoform sequences using primers designed to exploit nucleotide sequence differences between SNAP-25a and 25b (see Table 2). Panel A, Calibration curve of quantitative RT-PCR performed using primers sets on SNAP-25 isoform cDNA. Ct values (triplicates ± SEM) obtained for each primer/cDNA plasmid pair were plotted versus the amount of DNA template on a log scale to demonstrate the linear relationship between amplification and DNA input. Robust and equal amplification of each SNAP-25 isoform cDNA was detected only with the appropriate primer set and corresponding plasmid (SNAP-25a: 25a/25a, blue closed squares; and SNAP-25b 25b/25b, orange open squares). Only non-specific, negligible amplification (CT values >30) was obtained from non-corresponding primer/plasmid sets (e.g. 25a/25b, green triangles; 25b/25a red circles). Panel B, RT-PCR performed on RNA of transfected cells. COS7 cells (1.5 × 105 cells/well of a 12 well plate) were transfected with equivalent molar amounts (~1 μg/well) pCDNA3 expression plasmids bearing SNAP-25 isoforms , or the empty vector (pCDNA), using Lipofectamine (Invitrogen, Carlsbad CA, USA). cDNA prepared from RNA (1 μg) extracted from the transfected or untransfected (Untrans) control cells was amplified using the indicated isoform specific reverse primer, either individually or together (25a+25b), by conventional end-point reverse transcriptase PCR (40 cycles, see Methods). Amplification using a primer set to S12 rRNA protein transcripts served as a positive control. The size of the isoform specific amplicons (SNAP-25a 149 bp; SNAP-25b, 176 bp) is due to the different positions of isoform-specific reverse primers relative to the common forward primer, panSNAP25. The lack of a band in the mismatched primer/template lanes reflects the specificity of the PCR reaction. Panel C, Quantitative RT-PCR using total RNA preparations from cortex of wild type (WT, white bar) and SNAP-25a overexpressing knockin mutant (Snap25tkneo, black bar) mice. The relative overexpression of SNAP-25a transcripts in the homozygous mutant (Tkneo/Tkneo) samples, expressed as ΔΔCt (25a/25b) normalized to wild type (WT), was calculated from the SNAP-25 isoform amplification relative to β-actin (ΔCt value) and then deriving the relative ratio of their amplification rates (ΔΔCt). The ratio of SNAP-25a to SNAP-25b (e.g. 25a/25b) obtained from the mutant was then normalized to wild type (set at 1.0) to obtain a fold increase of SNAP-25a expression in these mutant mice. This increased level of SNAP-25a isoform transcripts in the mutant cortex RNAs, seen in both young (P24) and adult (P124), is consistent with values previously reported for these mice  confirming the specificity of each primer set and their use in assaying the expression of SNAP-25 isoforms in brain tissue.