Real Time PCR is a fast and efficient way to quantitate DNA product and is routinely used by labs worldwide. The Real Time PCR chemistry most commonly used is SyBr green, a fluorescent compound that registers the relative amount of cDNA amplicon by binding double-stranded DNA. Thus, signal increases as more dsDNA is produced during the PCR reaction. Applied Biosystem's TaqMan Gene Expression Assays primer/fluorescent probe sets are an alternative chemistry to SyBr green. In these assays, primers amplify a small region of cDNA to which FAM-labeled fluorescent complimentary strand probes bind and emit a fluorescent signal as the reaction proceeds. Unlike SyBr green that can produce a non-specific signal if primer dimers are formed, the ABI TaqMan Gene Expression Assay gives specific and sensitive results because the fluorescent probe binds ONLY to the amplified product. Contamination by genomic DNA is negated because, wherever possible, primer sets span exons. All primer sets utilize universal cycling conditions that omit the need for optimization and allow the running of many primer sets simultaneously. Indeed, ABI even replaces primer/ probe sets that don’t work.
In total, over 400,000 TaqMan Gene Expression Assays are available and include primer/ FAM-labeled probe sets for human, murine and rat genes. In most cases, multiple primer/ probe sets are available for each gene, however only “Inventoried” genes (i.e. those followed by a green box on the online catalogue) are verified specific for the gene of interest. Inventoried Assays are provided in single tubes of 20x reaction mix that are sufficient for 250-20ul reactions. “Non-inventoried” assays are available in 360 reaction lots. Common house-keeping genes are available in reaction sets of 1000 and can be purchased with VIC or FAM-labeled probes. If requested, ABI can also design custom gene expression assays to search for specific regions of genes. On the down side, ABI does not provide exact primer/ probe sequences. However, the information sheet and CD that are sent with the assays allow the researcher to get an approximate location of the region that is amplified.
In my experience, the ABI TaqMan Gene Expression Assays are a great way to quantitate specific gene products in singleplex experiments. I purchased twelve FAM-labeled TaqMan Gene Expression Assays and a VIC-labeled endogenous control gene to analyze my gene products via multiplex (control and gene of interest in a single reaction). When multiplex analysis is used, ABI recommends that the primers be tested alone and in combination prior to large-scale analysis. Indeed, I found that in all but one primer set where multiplex PCR was performed, my triplicate reactions were between 0.5 and 2.5 cycles apart and that the gene products were amplified up to four cycles later compared to singleplex experiments. Singleplexing, however, provided very tight replicates, less than 0.5 cycles apart.
Vanta Jokubaitis
Graduate Student
Stem Cell Research Laboratory
Peter MacCallum Cancer Centre
Melbourne, Australia