The Applied Biosystems Assays-on-Demand™ Gene Expression products are a comprehensive collection of pre-designed primer and probe sets for quantitative real-time PCR gene expression studies. Each assay is designed using Applied Biosystems bioinformatics design pipeline, using both public and private genome databases for increased specificity and optimal assay design. The vast majority of the tests are designed to assay over large introns therefore contaminating genomic DNA will not be amplified. The test is based on a two-step reverse transcription-polymerase chain reaction (RT-PCR) in which the PCR step is coupled with a 5´fluorogenic nuclease assay.
Each pre-formulated assay comes in a 20X mix and contains two unlabeled PCR primers and a FAM™ dye-labeled gene-specific TaqMan® MGB probe. Each assay is tested on pooled human cDNA to verify the performance. They also supply compact disk containing assay information file with primers and probe sequences. Researcher only needs to add TaqMan® Universal PCR Master Mix and cDNA sample to generate quantitative gene expression data on Applied Biosystems ABI PRISM® 7900HT, 7700, or 7000 Sequence Detection Systems. Compared to do-it-yourself methods, these pre-made tests are fast to implement, require virtually no troubleshooting, and eliminate weeks of extra work.
In order to find out whether an assay is available for a particular gene one needs to know gene name, or NCBI RefSeq number, or gene family. Initially I accessed the database of available assays through Celera Discovery System for which I was a paid subscriber. But when I recently looked at the Applied Biosystems website I found that anyone can register there free and search the database directly. Their website now lists more than twenty one thousand rigorously tested assays for human transcripts, and thousands more for mouse and rat transcripts.
I used Assay-On-Demand TM gene expression product to study quantitative expression of several human genes. We purchased and used similar test for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Hs_999999905_m1) as a control. In a single tube I combined 20X assay mix, 2X Tagman® Universal PCR master mix, DEPC water and cDNA. The 7900HT Sequence Detection System (Applied Biosystems) was used to collect real-time fluorescent signal. All the assays I used required no troubleshooting and worked beautifully first time with an excellent reproducibility.
Although a number of projects would greatly benefit from using this system, which produces fast results, it is not suitable for all projects. It is not suited for detecting splice-variants, for example. In addition, one needs to realize that because these assays are pre-designed and pre-made they cannot be modified or changed to suit any specific needs.
In short, Assays-on-Demand™ is a fast, easy and very convenient product for testing of quantitative expression of known gene.
Yelena Bykhovskaya, PhD
Research Scientist I
Molecular Hematology Laboratory
Cedars-Sinai Medical Center