Applied Biosystems’ TaqMan® Gene Expression Assays (GEAs) are a straightforward and reliable way of accurately quantifying virtually any gene in the human, rat, or mouse (among other) genomes by real-time quantitative PCR (qPCR). Each assay is optimized for standardized PCR conditions and supplied as a 20X mixture containing primers and probe. Amplicons are designed to cross exon-exon junctions and are about 150 base pairs, so probes are gene-specific and will not amplify genomic DNA. In addition, the probe is detected only upon amplification of your specific gene product. All GEA probes have a FAM reporter dye at the 5’end of the Taqman® MGB® (Minor Groove Binding) probe and a nonfluorescent quencher at the 3’ end of the probe. Simply search their database for your species and your gene/gene region of interest.
I have used the TaqMan® GEAs for relative quantification of several rat genes by qPCR on an ABI 7000 over the last several months. I have been using total RNA (then converted to cDNA) isolated from rat heart. I quickly became tired of optimizing, PCR efficiency testing, redesigning primers, and unreliable results (i.e. nonspecific amplification) with SYBR® Green-based qPCR, so switched over to TaqMan® GEAs. I am using GEAs as a second, more sensitive method of validating microarray data. So far, I have used the assays to characterize several genes (mostly rare transcripts) involved in mitochondrial biogenesis.
The TaqMan® probes are gene specific and easy to use in this 20X format: All I do is combine my cDNA template and PCR master mix; I perform singleplex reactions. Since I am quantifying rare transcripts, I usually use approximately 1 ug of cDNA per 20 ul singleplex reaction and normalize data with an 18S rRNA endogenous control. Typically, it takes me about 1-2 hours to set up a 96-well plate which includes 3 genes and 10 different cDNA samples. The entire PCR run takes about 2 hours to complete and uses the same PCR cycling conditions for each assay. After the PCR run is complete, I export the data into an Excel file and calculate the average threshold cycle (Ct) for three technical replicates per sample. From the average Ct values given for each sample, I then calculate the delta (dCT) and delta-delta (ddCT) values to get the fold differences in gene expression.
Although the GEAs are expensive, they are well worth the cost and actually end up saving you time and money as compared to other chemistries which require optimization. The pre-made, inventoried 20X assays are a little cheaper than the custom assays but you will have to wait 1-2 weeks for them to arrive if they are not in stock (although you get more reactions with the custom assays). Some standardization may also be required if you want to ensure accurate results, so it is always a good idea to run a validation experiment when using the ddCt (aka ∆∆Ct or Comparative Ct) method to ensure your calculations of fold change are valid. The validation experiment is simply running a standard curve (regression line) between dCt and your log input of cDNA, and this tells you if the PCR efficiency of your target gene and your reference gene are approximately equal. PCR efficiencies should always be close to 100% because amplification is exponential.
Darcey L. Klaahsen
Research Lab Manager
Kansas State University
Department of Human Nutrition