Primer Express software does not select probes with a G on the 5´ end. The quenching effect of a G base in this position will be present even after probe cleavage. This can result in reduced normalized fluorescence values (DRn), which can impact the performance of an assay. Having G bases in positions close to the 5´ end, but not on it, has not been shown to compromise assay performance. Another empirical observation is that probes with more C than G bases will often produce a higher DRn. Since Primer Express software does not automatically screen for this feature, it must be checked manually. If a probe is found to contain more G than C bases, the complement of the probe selected by Primer Express software should be used, ensuring that a G is not present on the 5´ end.
The last five bases on the 3´ end of the primers should contain no more than two C and/or G bases, which is another factor that reduces the possibility of non-specific product formation. Under certain circumstances, however, such as a G/C-rich template sequence, this recommendation may have to be relaxed to keep the amplicon under 150 base pairs in length. It should, however, be followed as often as possible, and even when it is not possible, primer 3´ ends extremely rich in G and/or C bases should be avoided.
All quantitative assays designed using Applied Biosystems’ guidelines can be run using the same universal thermalcycling parameters. This eliminates any optimization of the thermal cycling parameters and means that multiple assays can be run on the same plate without sacrificing performance. This benefit is critical when combining two assays into a multiplex TaqMan assay system, in which the option to run the assays under different thermal cycling parameters is not available. Table 2 shows the universal thermal cycling parameters for quantitative TaqMan or SYBR Green I assays when using DNA or cDNA as the substrate.
F. Sample Preparation
The user is responsible for setting up their own reactions. The samples should be supplied to the DNA Facility in tubes or plates that are ready to be placed into the instrument. In other words, the user is responsible for acquiring the reaction reagents, primers, probe, and reaction vessel. Applied Biosystems TaqMan reagents can be obtained from the Enzyme Core at the Hybridoma Facility (2nd Floor EMRB). The DNA Facility will be responsible for setting up the instrument, data collection, and preliminary data analysis.
Results will be available in two forms. The first is a hard copy of the Experimental Report as provided by the Sequence Detection System Analysis Software v. 1.7. The output is presented in a tabular form in which each row corresponds to information generated for each well position. For each well, information regarding the sample name, CT value, and quantity or copy number is provided. If a standard curve was run, its slope, fit (R), and y-intercept is provided in the header information. Alternatively, these data can be provided in an electronic format as a Microsoft Excel workbook file. Additional data analysis, such as DDCT calculations, is the responsibility of the user. However, core personnel are available to assist and/or advise users in performing these additional calculations.
- Real-time PCR review article (2000) (RealtimeRevarticle.pdf)
- Real-time PCR review article (2002) (Bustin_realtimereview_2002.pdf)
- Real-time PCR overview (realtimeoverview)
- Basics of using Real-time PCR (realtimePCRbasics)
- Gene quantification calculations (genequant.)
- Relative quantitation of gene expression (Compar_Anal_Bulletin2.)
