ProbeLibrary: A new method for faster design and execution of quantitative real-time PCR

ProbeLibrary, a fast, specific and flexible format for quantitative real-time PCR, is described. The ProbeLibrary concept is based on the fact that just 90 short probes provide transcriptome-wide coverage in most organisms. These short probes are highly specific and possess the high melting temperature (Tm) required for real-time PCR owing to the incorporation of locked nucleic acid (LNA). The probes are dual-labeled fluorogenic probes that are used in standard real-time PCR protocols, with standard instrumentation. ProbeLibrary probes are used in assays designed using free, web-based assay design software, termed ProbeFinder. In seconds, ProbeFinder designs highly specific intron-spanning assays for a target transcript. The system allows multiple design options, including designs for transcript variants and gene family members. Assay design success rate is 96%. This combination of extremely fast assay design and instant availability of the probes leads to faster implementation of real-time PCR assays.