Nucleic Acids Research Group 2003 Study: Evaluation of Multiple Taqman ® Assay Designs: What Did We Learn from Looking at 32 Assays Made for Mouse IFNγ?

Le résumé fourni par la source

The Nucleic Acids Research Group (NARG) of the Association of Biomolecular Resource Facilities (ABRF) invited researchers to participate in an empirical study to define the parameters required to make an optimal 5‘nuclease (Taqman¨) real-time PCR assay. New assay design can be one of the major rate-limiting steps to those new to this technology in rapidly acquiring data from their genes of interest. Although a large number of pre-made assays can be purchased from Applied Biosystems and Qiagen, it is much more cost effective to make your own assays if a large number of samples need to be run with each assay. There are general guidelines available concerning assay design. However, exactly how important each of these parameters are has not been studied in an empirical manner. Further, there may be as yet unknown factors that should be taken into account during assay design. The purpose of this study was to give a large number of investigators an opportunity to design what they feel will be an optimal primer/probe set for a common transcript and then have them tested empirically for efficiency. Each participant was asked to provide the sequence of a pair of primers and a probe within a slightly reduced coding region of the mouse IFNg transcript. Members of the NARG synthesized the primers and probes, and tested the reagents for each assay using a recombinant plasmid containing a partial mouse IFNg cDNA as template. The results for each assay will be posted on the ABRF web site and published. Entries have been identified by a user designated code and are completely anonymous.

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