TaqMan^® Gene Expression

Introduction

Applied Biosystems developed TaqMan^® Gene Expression Assays, a genome-wide collection of quantitative, standardized assays for gene expression analysis by real-time quantitative RT-PCR. The TaqMan^® Assay utilizes the 5' nuclease activity of Taq DNA polymerase to cleave a fluorescently labeled probe (FAM^TM-labeled MGB).

These assays are a family of more than 700,000 primer/probes sets, which have been pre-designed by using a state-of-the-art, extensively validated (~20,000 assays were functionally tested) bioinformatics pipeline. Assays for quantification of gene-encoded transcripts are available for Homo sapiens, Mus musculus, Rattus norvegicus, Drosophila melanogaster, Arabidopsis thaliana, Canis familiaris, Caenorhabditis elegans, and Macaca mulatta.

TaqMan^® Gene Expression Assays Design Pipeline

Automated Primer/Probe Design Pipeline.

This figure highlights the design pipeline and rigorous quality checks that the TaqMan^® Gene Expression Assays must pass.

Methods

• Source of transcripts

  • The assays were designed based on gene-encoded transcripts obtained from the NCBI Reference Sequence Project database (RefSeq), the best known, most highly curated set of non-redundant transcripts in the public domain.

  • Transcript pre-processing

  • Repetitive and low complexity regions in each transcript are masked (i.e., nucleotides are replaced by "N").

  • The masked transcripts are mapped to the NCBI genome assemblies with an Applied Biosystems proprietary alignment tool. The positions of each exon-exon boundary are marked for each multi-exon transcript; single-exon transcripts are identified as such.
  • All known SNPs sites are masked after performing a BLAST (Basic Local Alignment Tool) analysis against the NCBI and Celera SNP database.

  • Assay design

  • Each assay includes a single FAM^TM dye-labeled TaqMan^® probe with a minor groove binder (MGB) moiety and two unlabeled oligonucleotide primers.

  • These components are designed with a program called TaqExpress, which takes into account optimal melting temperature (T_m) requirements, GC-content, buffer/salt conditions, oligonucleotide concentrations, secondary structure, optimal amplicon size, reduction of primer-dimer formation, etc.
  • In case of multi-exon transcripts, probes are placed at each exon-exon boundary to ensure that the primers bind in two distinct exons and that the fluorescent signal is only generated from correctly spliced templates.

  • In silico quality scoring

  • Transcript BLAST scoring

    Determining the degree of homology, through BLAST, between the assay and other related transcripts. A penalty is assigned if an assay detects any closely homologous transcripts other than the intended target.

  • Genome BLAST scoring

    A penalty is assigned if an assay hits other physical locations on the genome in addition to the location of the target gene.

  • Intron size scoring

    A penalty is assigned when the assay is designed across an exon-exon boundary that spans a small intron (i.e., <2 kb).

  • Only high quality assays are selected for manufacture and made available on the website.

• Relational database for 5' nuclease assay designs (TaqDB)

  • Information about transcripts, assays, global relationships between transcripts and assays, exon-intron structure, in silico QC, manufacturing order status, and analytical QC data determined in the manufacturing process are maintained in a relational database at Applied Biosystems. Data from expression studies in select RNA tissue pools are also stored in this database.

  • Remapping

  • Because transcript databases change over time (new transcripts are added, current transcripts may be revised or removed), Applied Biosystems regularly remaps the assays to NCBI transcript database.

Resources

» TaqMan^® Gene Expression Assays

» TaqMan^® Gene Expression Assays Protocol (.pdf)

» TaqMan^® Gene Expression Assays Design Pipeline (.pdf)

» Applied Biosystems

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