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. 2006 Jul 19;34(12):3421–3433. doi: 10.1093/nar/gkl390

Figure 3.

Figure 3

Product-specific real-time PCR. (a) A schematic representation of the underlying design of the quantitation method. Authentic and latent mRNAs are specifically hybridized to PCR primers that flank the junction between a sequence upstream of the respective 5′ splice site and a sequence in the downstream exon. The specificity for the pre-mRNA stems from the location of the sense primer downstream of the latent site. Wide box, exon; line, intron; narrow box, latent exon. (b) Gel electrophoretic analysis of RT–PCR products from transcripts of wild-type CAD2 (lane 2) and the stop-codon-less CAD2-Mut1 (lane 3) constructs. Bands corresponding to precursor (428 bp), authentic (203 bp) and latent (328 bp) CAD fragments amplified with primer pair a/b are indicated by schematic drawings on the right. (c) RNA isolated form cells transfected with CAD2 (lanes 2–4) or with CAD2-Mut1 (lanes 5–7) were analyzed with the specific primer pairs as indicated: The precursor-specific primer pair (lanes 2 and 5); the authentic-specific pair (lanes 3 and 6) and the latent-specific pair (lanes 4 and 7). PCR products were analyzed on a denaturing 7.5% polyacrylamide gel.