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TaqMan® Gene Expression Products


Life Technologies offers more than 1.3 million predesigned TaqMan® Gene Expression Assays covering 23 species, including some pathogens, the most comprehensive set of real-time PCR assays available. …

Life Technologies offers more than 1.3 million predesigned TaqMan® Gene Expression Assays covering 23 species, including some pathogens, the most comprehensive set of real-time PCR assays available. All TaqMan® Gene Expression Assays have been designed using validated bioinformatics pipeline, and run with the same PCR protocol, eliminating the need for primer design or PCR optimization.

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  • 1. Product Guide TaqMan® Gene Expression Assay Products TaqMan Gene Expression Assays ® Product Guide
  • 2. Applied Biosystems TaqMan ®Gene Expression Assays SolutionsApplied Biosystems offers the largest family of products to meet your quantitativegene expression needs: from off-the-shelf gene-specific probe and primer sets toCustom TaqMan® probes and primers manufactured to your desired sequences,and everything in between. All products use TaqMan® probe-based chemistry andare designed for use on the suite of Applied Biosystems Real-Time PCR Systems—together the gold standard in quantitative gene expression offering the greatestsensitivity, specificity, reproducibility, and the broadest dynamic range. Table of Contents TaqMan® Gene Expression Assays 2 Custom TaqMan® Gene Expression Assays 4 TaqMan® Endogenous Controls 5 TaqMan® Low Density Array 7 Custom TaqMan® Probes and Primers 9 TaqMan® MicroRNA Assays 10 Applied Biosystems Real-Time PCR Systems 12 Product Specification Comparison Back CoverWhich TaqMan® TaqMan® Custom TaqMan® TaqMan® Custom TaqMan®Gene Expression Product Gene Expression Assays Gene Expression Endogenous Probes andIs Right for You? Inventoried Non-Inventoried Assays Controls PrimersConvenience of fully-formulatedassay (premixed probe and primers)    No specific preference onassay location within a gene Specific location withina transcript is key   FAM™ dye labelis fine     Need a specificreporter dye label  Prefer to use anApplied Biosystems design    Prefer to use my ownsequence target  Prefer to design my ownprobe and primers Non-universalprobe-primer concentration pg. 1 |
  • 3. TaqMan® Gene Human Assays strains. By avoiding areas in the geneExpression Assays Over 100,000 gene expression assays transcripts of known polymorphisms,• Gene-specific TaqMan® probe are available for all known human we design only strain-neutral gene and primer sets for quantitative genes. These include genes in the expression assays. gene expression studies in human, public domain with associated RefSeq mouse, rat, Arabidopsis, and transcripts (NCBI Reference Sequence Arabidopsis and Drosophila Assays Drosophila species project database: http://www.ncbi. Over 130,000 assays have been, the mammalian designed for most known genes for• Convenient single-tube format gene collection (MGC), and GenBank® Arabidopsis thaliana and Drosophila• Universal cycling conditions melanogaster. Similar to the assays for database. A minimum of one assay (probe and primer set) per RefSeq other species, these assays have beenTaqMan® Gene Expression Assays transcript is available as an inventoried, designed against the public RefSeqare a comprehensive collection of off-the-shelf product currently num- database. The collection includes assaysover 600,000 pre-designed probe and bering > 21,000 assays. The complete for nearly every known transcript forprimer sets that let researchers quickly collection includes assays for nearly each organism, and all assays areand easily perform quantitative gene every exon junction in all known available on a made-to-order basis.expression studies on human, mouse,rat, Arabidopsis, or Drosophila genes. human genes in the public domain Comprehensive CoverageEach gene expression assay consists of covering most probes on the Applied and Selectiona FAM™ dye-labeled TaqMan® MGB Biosystems Expression Array System. Not only have we designed an assayprobe and two PCR primers formu- for every gene, but also for multiple Mouse and Rat Assayslated into a single tube. Every assay locations across each gene transcript. Over 200,000 mouse and rat assaysis optimized to run under universal More than 600,000 high-quality assay have been designed for all known genes.thermal cycling conditions with a final designs are available for human, mouse, As with our human assays, at least onereaction concentration of 250 nM rat, Arabidopsis, and Drosophila genes assay per RefSeq transcript has beenfor the probe and 900 nM for each on a made-to-order basis. This vast manufactured and is available from ourprimer. This streamlined approach and selection allows researchers to select the inventory. High quality assay designscomprehensive assay selection enables specific location on a given transcript for all other genes are also availablea convenient, standardized process they wish to detect. For instance, on a made-to-order basis, asfor quantitative gene expression. microarray researchers that may prefer non-inventoried assays. a 3 bias in their TaqMan® probe and Strain-Neutral Mouse and Rat Assays primer sets will be able to select from The assay design process yields strain- robust, pre-designed TaqMan® Gene neutral mouse and rat gene expression Expression Assays. Additionally, assays. Polymorphisms are the cause researchers performing RNAi studies of most sequence variability between can choose multiple assays per gene to validate their knockdown results. TaqMan® Gene Expression Assays are delivered with a compact disc containing an electronic assay information file. | pg. 2
  • 4. State-of-the-Art AssayDesign BioinformaticsAll assays are designed using AppliedBiosystems sophisticated bioinformaticspipeline, customized for either thehuman, mouse, rat, Arabidopsis, orDrosophila genome. This pipelineconsists of three main steps:Step OneBoth public and Celera sequence data areused to identify the optimal probe andprimer locations. This process consists of:• Mapping transcripts to the genome to identify exon boundaries• Masking sequence discrepancies between public and Celera data• Masking sequence repeats The Applied Biosystems online catalog map view graphically represents the assay’s physical location on the gene, and which transcript(s) each assay detects.• Masking known SNPs from both public and Celera databases catalog and are available on a made-to- to each of the eight sample ports andStep Two order basis. Our graphical map viewer run on an Applied Biosystems 7900HTProprietary software algorithms shows each assay’s location on the Fast Real-Time PCR System. For moregenerate probe and primer designs for gene, to help determine which assay information, see the “TaqMan® Lowthe locations identified above. These is most appropriate. Density Array” section on page 7.algorithms include optimal design Choice of Delivery Formats A TaqMan® Genomic Assay Catalogparameters, such as %GC content, Tm, Applied Biosystems delivers the assays CD is also available. This off-line versionamplicon length, and low secondary in either a tube-format or TaqMan® Low of our catalog allows you to search andstructure, to ensure high amplification Density Array-format (using inventoried select our assays without an Internetefficiency. Where possible, designs span assays only). The TaqMan® Low Density connection. Download a version todayan exon-exon junction, eliminating the Array is a 384-well micro fluidic card at or request a copypossibility of detecting genomic DNA. that streamlines reaction set-up time, from your sales representative.Step Three eliminates the need for liquid handling Convenient Assay Ordering OptionsIn silico QC ensures each assay is robotics, and provides standardization Both our online catalog and CDspecific to the gene for which it across multiple users and/or multiple catalog enable searching for yourwas designed (i.e., the assay will not labs. This format is ideal for analyzing target of interest by:detect sequences from other genes, many samples across a fixed number ofor pseudo-genes). Each assay design targets, such as for biomarker screening. • Gene nameis processed through a quality scoring The TaqMan® Array arrives ready to • Accession numbersystem and one high scoring, gene- use, with your selected TaqMan® Gene (NCBI RefSeq ID number, etc.)specific assay design is sent to our Expression Assays preloaded into each of • Gene family, or functional categoriesstate-of-the-art manufacturing facility. the 384 reaction wells. Simply add 100 and groups (kinase, transcriptionAll designs meeting our scoring criteria µL sample mix (sample cDNA and factor, ion channel, cytokine, etc.)are also displayed in our online TaqMan® Universal PCR Master Mix)pg. 3 |
  • 5. Custom TaqMan® GeneExpression Assays• Any species or organism• Target sequence of your choice• Convenient single-tube formatCustom TaqMan® Gene ExpressionAssays are available for any species,any splice variant, or any novel gene.Simply download our free File BuilderSoftware to format and submit yourtarget sequence. File Builder Softwarecan be downloaded from our Web site( software easily guides you throughthe ordering process from selectingthe assay size, formatting your targetsequence to identify the location ofthe probe, and submitting your ordervia e-mail. Custom TaqMan® Gene Expression Assays are delivered ready-to-use, along with the probe and primer sequences you designed.File submissions are done in a secureformat. Your target sequences andthe associated assays that are designed 96-position rack designed to accommo- into a single 20X mix. Designed to runare kept confidential. With Custom date standard liquid handling robotics under universal conditions for two-stepTaqMan® Gene Expression Assays, and fit seamlessly into automated, high- RT-PCR, TaqMan® Gene Expressionyou benefit from Applied Biosystems throughput laboratory processes. Each Assays are simple to use. Just addproprietary software algorithms for order of assays also includes a compact TaqMan® Universal PCR Master Mixprobe and primer design, which enable disc with an assay information file that (with or without AmpErase® UNG)you to obtain optimal assays for each includes the assay ID numbers, detector and your cDNA sample to generatetarget sequence. Assays are delivered in names, reporter dye, and quencher sensitive, reproducible, and trulya single-tube, ready-to-use format, along information for easy uploading into quantitative gene expression datawith the probe and primer sequences a LIMS or sequence detection on any Applied Biosystems Real-Timedesigned from your submitted sequence. system software. PCR System.Automation-Compatible to Accelerate A Simple, Standardized Solution Compared to do-it-yourself methods,High-Throughput Applications for Quantitative Gene Expression TaqMan® Gene Expression Assays andBoth TaqMan® Gene Expression Assays TaqMan® Gene Expression Assays and Custom TaqMan® Gene Expressionand Custom TaqMan® Gene Expression Custom TaqMan® Gene Expression Assays eliminate weeks or even monthsAssays come pre-formulated in a single, Assays are built on our 5 nuclease of probe and primer design, formula-2D-barcoded tube with an easy-to-read chemistry and consist of a FAM™ tion, and testing.label. The single-tube format requires dye-labeled TaqMan® MGB probe For more information on this product,fewer set-up and pipetting steps to (250 nM, final concentration), and visit or checkassemble reactions, allowing you to two unlabeled PCR primers (900 nM with your local Applied Biosystemseasily scale your throughput. Assay each, final concentration). All compo- representative.tubes are shipped in a 1D-barcoded nents are QC tested and formulated | pg. 4
  • 6. A Simple, Standardized Solution Choosing the Right for Quantitative Gene Expression Endogenous Control Each endogenous control is built Endogenous controls can normalize on our 5 nuclease chemistry and is the expression levels of target genes by offered in a choice of two different correcting differences in the amount reporter dyes and two quenchers: of cDNA that is loaded into PCR reac- • A FAM™ dye-labeled TaqMan® MGB tion wells. For best results, verify that probe (250 nM, final concentration) the endogenous control is consistently and two unlabeled PCR primers expressed in the sample set to be tested. (900 nM each) Endogenous control expression must be uniform across all samples in the study. • A VIC® dye-labeled TaqMan® MGB For multiplexing, ensure that the gene probe (250 nM, final concentration)Each endogenous control is built on our 5 nuclease expression level of the endogenous con- and two unlabeled PCR primerschemistry and is available in a number of reporter trol is greater than that of the target.dye/quencher formats. (150 nM each—primer limited) • A VIC® dye-labeled TAMRA™ Multiplex vs. Singleplex PCRTaqMan® Endogenous Controls dye-labeled probe (250 nM, final All TaqMan® Endogenous Controls• Optimized, pre-formulated, concentration) and two unlabeled that contain probes labeled with the ready-to-use control assays PCR primers (150 nM each— VIC® reporter dye are primer limited.• Cost-effective gene expression primer limited) This allows multiplexing of TaqMan® quantitation in human, mouse, Endogenous Controls with target rat, Arabidopsis, Drosophila, All components are QC tested, gene expression assays, provided that and eukaryotes formulated into a single 20X mix, and the control gene is more abundantly functionally tested. Designed to run expressed than the target gene. All• Choice of FAM™ or VIC® dye labels under universal conditions for two-step TaqMan® Endogenous Controls thatApplied Biosystems TaqMan® RT-PCR, our TaqMan® Endogenous contain probes labeled with the FAM™Endogenous Controls are a collection Controls are simple to use. Just add reporter dye are not primer limitedof pre-designed probe and primer TaqMan® Universal PCR Master Mix and are not intended for multiplexing.sets that can be used to normalize (with or without AmpErase® UNG) and your cDNA sample to generate Complementary Productsthe amount of sample RNA or DNA sensitive, reproducible, and truly TaqMan® Endogenous Controls areadded to a reaction. For the quantita- quantitative gene expression data intended to be used with:tion of gene expression, deciding upona specific control can be difficult, even on ABI PRISM® 7000 and 7700 • TaqMan® Gene Expression Assayswhen detailed information about the Sequence Detection Systems, • Custom TaqMan® Gene Expressionbiological system is available. This Applied Biosystems 7300 and 7500 Assayscan result in trial and error to identify Real-Time PCR Systems, and Applied • Custom TaqMan® Probes and Primersan appropriate control, leading to Biosystems 7500 and 7900HT Fastproject delays and increased costs. Real-Time PCR Systems. Compared Online OrderingApplied Biosystems offers endogenous to do-it-yourself methods, our Order from our line of TaqMan®controls for the most commonly used TaqMan® Endogenous Controls Endogenous Controls through thecontrol genes in human, mouse, rat, deliver a complete quantitation Applied Biosystems store atArabidopsis, Drosophila, and any solution and eliminate weeks or http://store.appliedbiosystems.comeukaryotic species. The assays are even months of assay design, formulation, and testing. For more information on this product,designed to let researchers quickly check with your local representative orand easily identify and run the best visit www.allgenes.compossible endogenous control fortheir gene expression 5 |
  • 7. TaqMan® Endogenous Controls Number of Dye/Quencher Part Number Primer Limited Conc. Reactions, 20 µLEukaryotic 18S rRNA VIC®/MGB 4319413E Y 20X 2,500 VIC /TAMRA ® ™ 4310893E Y 20X 2,500 FAM™/MGB 4333760T N 20X 125 FAM™/MGB 4333760f N 20X 500Human ACTB (beta actin) VIC®/MGB 4326315E Y 20X 2,500 VIC®/TAMRA™ 4310881E Y 20X 2,500 FAM /MGB ™ 4333762T N 20X 125 FAM™/MGB 4333762F N 20X 500Human B2M (beta-2-microglobulin) VIC®/MGB 4326319E Y 20X 2,500 VIC®/TAMRA™ 4310886E Y 20X 2,500 FAM™/MGB 4333766T N 20X 125 FAM /MGB ™ 4333766F N 20X 500Human GAPD (GAPDH) VIC®/MGB 4326317E Y 20X 2,500 VIC®/TAMRA™ 4310884E Y 20X 2,500 FAM /MGB ™ 4333764T N 20X 125 FAM™/MGB 4333764F N 20X 500Human GUSB (beta glucuronidase) VIC /MGB ® 4326320E Y 20X 2,500 VIC®/TAMRA™ 4310888E Y 20X 2,500 FAM™/MGB 4333767T N 20X 125 FAM /MGB ™ 4333767F N 20X 500Human HPRT1 VIC®/MGB 4326321E Y 20X 2,500 VIC®/TAMRA™ 4310890E Y 20X 2,500 FAM™/MGB 4333768T N 20X 125 FAM™/MGB 4333768F N 20X 500Human PGK1 (phosphoglyceratekinase 1) VIC /MGB ® 4326318E Y 20X 2,500 VIC®/TAMRA™ 4310885E Y 20X 2,500 FAM /MGB ™ 4333765T N 20X 125 FAM™/MGB 4333765F N 20X 500Human PPIA (cyclophilin A) VIC®/MGB 4326316E Y 20X 2,500 VIC®/TAMRA™ 4310883E Y 20X 2,500 FAM™/MGB 4333763T N 20X 125 FAM /MGB ™ 4333763F N 20X 500Human RPLO (large ribosomal protein) VIC®/MGB 4326314E Y 20X 2,500 VIC®/TAMRA™ 4310879E Y 20X 2,500 FAM /MGB ™ 4333761T N 20X 125 FAM™/MGB 4333761F N 20X 500Human TBP (TATA-box binding protein) VIC /MGB ® 4326322E Y 20X 2,500 VIC®/TAMRA™ 4310891E Y 20X 2,500 FAM™/MGB 4333769T N 20X 125 FAM /MGB ™ 4333769F N 20X 500Human TFRC (CD71) (transferring receptor) VIC®/MGB 4326323E Y 20X 2,500 VIC®/TAMRA™ 4310892E Y 20X 2,500 FAM™/MGB 4333770T N 20X 125 FAM™/MGB 4333770F N 20X 500Mouse GAPD (GAPDH) VIC /MGB ® 4352339E Y 20X 2,500Mouse ACTB (beta actin) VIC®/MGB 4352341E Y 20X 2,500Rat GAPD (GAPDH) VIC /MGB ® 4352338E Y 20X 2,500Rat ACTB (beta actin) VIC®/MGB 4352340E Y 20X 2,500Table 1. | pg. 6
  • 8. TaqMan® Low Density Array assays to run on the TaqMan® Array researchers in the field. All assays are• TaqMan® performance in a low (for selection, visit designed to genes with known implica- density array format TaqMan® Arrays are available in nine tions in immune response. For conven-• Micro fluidic technology provides different formats, covering 12, 16, 24, ience, these TaqMan® Array Immune access to 384-well format without 32, 48, 64, 96 (2 choices), and 384 Panels are available in packs of ten. liquid-handling robotics assays per array (Table 3). TaqMan® Arrays are designed for use on the The endogenous control panel allows• Standardization enables screening flexible Applied Biosystems 7900HT researchers to quickly identify the opti- of multiple samples against selected Fast Real-Time PCR System. mal gene(s) to use as endogenous con- gene panels trols for each gene expression study. Ideal Screening Technology Eleven TaqMan® Endogenous ControlsThe TaqMan® Low Density Array TaqMan® Low Density Arrays are ideal for human, plus five additional TaqManis an easy-to-use micro fluidic card for screening applications such as: Gene Expression Assays (identifiedfor real-time PCR. The micro fluidic as exhibiting minimal variation intechnology utilizes eight sample-loading • Biomarker screens gene expression across sixteen humanports, each connected to 48 reaction • Toxicology screens tissues), are pre-loaded in the TaqMan®chambers, streamlining the reaction • Target class screens Array in triplicate. Eight samples canset-up process and eliminating the be run on each array. The TaqMan®need for liquid-handling robotics. • Pathway analysis Array Endogenous Control PanelsSimply load 100 µL of sample mix, • Disease set analysis are available in packs of two.(30 –100 ng of cDNA sample and50 µL of 2X TaqMan® Universal PCR In addition to Custom TaqMan Arrays, Applied Biosystems 7900HTMaster Mix), into each of the sample Applied Biosystems also offers two Fast Real-Time PCR Systemloading ports. Centrifuge briefly, seal versions of TaqMan® Low Density The TaqMan Array was designedthe TaqMan® Array, and run on the Array Panels—the TaqMan® Low expressly for the Applied BiosystemsApplied Biosystems 7900HT Fast Density Array Immune Panel and 7900HT Fast Real-Time PCR System,Real-Time PCR System. the TaqMan® Low Density Array a flexible, high-throughput, Real-Time Endogenous Control Panel. The PCR system that detects and quanti-TaqMan® Low Density Arrays immune panel contains 96 TaqMan tates nucleic acid sequences. Theare delivered with your choice of Gene Expression Assays pre-loaded in combination of the TaqMan Arrayinventoried TaqMan® Gene Expression quadruplicate across the 384 reaction and the 7900HT system helps makeAssays pre-loaded into each of the 384 wells. The assays were selected in high-throughput gene expressionreaction wells. Select from our collec- collaboration with the Immune research successful and easy.tion of more than 40,000 inventoried Tolerance Network, as well as otherTaqMan® Low Density Array # of Samples per Card Minimum OrderDescription # of Assays 1 Replicate 2 Replicates 3 Replicates 4 Replicates Quantities Part NumberFormat 12 11 + 1 control 8 20 4342247Format 16 15 + 1 control 8 20 4346798Format 24 23 + 1 control 8 4 20 4342249Format 32 31 + 1 control 4 20 4346799Format 48 47 + 1 control 4 2 20 4342253Format 64 63 + 1 control 2 20 4346800Format 96a 95 + 1 control 2 1 20 4342259Format 96b 95 + 1 control 2 1 20 4342261Format 384 380 + 1 control 1 50 4342265Immune Panel 96 2 1 10 4342510Endogenous Control Panel 16 8 2 43675637900HT TaqMan Low Density Array Upgrade* ® 4329012Includes: sample block, micro fluidic card sealer, custom buckets and adaptors, Getting Started guide, and chemical installation kit.*A compatible centrifuge is required but not supplied.Table 7 |
  • 9. Available TaqMan® Low Density Array Formats Format 12 (part number 4342247) Format 64 (part number 4346800) Format 16 (part number 4346798) Format 96a (part number 4342259) Format 24 (part number 4342249) Format 96b (part number 4342261) Format 32 (part number 4346799) Format 384 (part number 4342265) Format 48 (part number 4342253)Table 3. TaqMan® Low Density Arrays can be ordered in any one of these nine formats. | pg. 8
  • 10. Custom TaqMan® Probes will have a greater destabilizing effect Primer Express® Design Softwareand Primers on an MGB probe, resulting in better Applied Biosystems Primer Express®• Choice of dye labels, quenchers, discrimination. The shorter length also software is available to simplify the and synthesis scales offers greater design flexibility for all probe and primer design process.• Available for any species or organism real-time PCR applications. Primer Express® is available for individual users and in multi-user• For use in quantitative gene Applied Biosystems offers the tradi- packs. Please check our Web site at expression, SNP genotyping, other tional dual-labeled Custom TaqMan® for allelic discrimination applications, Probes with a TAMRA™ dye fluores- more details. and pathogen detection cent quencher as well. All TAMRA™ dye TaqMan® probes are HPLC And, if you prefer the convenienceWhen you know the exact sequences purified. of a pre-designed probe and primeryou need for your TaqMan® Probes set, check our vast online selectionand Primers, Applied Biosystems can A Selection of Reporter Dyes of TaqMan® Gene Expression andsynthesize them for you. As the market Applied Biosystems Custom TaqMan® SNP Genotyping Assays.leader in real-time PCR, our high- Probes can be ordered with a varietyquality custom products can be used of different reporter dyes to facilitate Other Fluorescent Dye-Labeledin all of your real-time and end-point your multiplexing applications. OligosPCR applications. These products offer Applied Biosystems also offers ayou the ideal in flexibility, whether you Synthesis Scales host of other custom oligo productsprefer to optimize your own reaction TaqMan® Custom Probes and Primers for use in many applications,formulation, or if you simply want are available in a choice of three standard including microsatellite-based linkageto buy in large quantities. sizes. Each includes a pre-defined quan- mapping, mutation detection, and tity of probe or primer to ensure thatOur Custom TaqMan® Probes and more. Please see our Web site, you get the same amount each time youPrimers are manufactured at three sites order and aren’t subject to variations inaround the world—the United States, for more details. synthesis yield. For larger synthesis scalesthe United Kingdom, and Japan, to on these products, please contact youroffer excellent delivery time. Order by Applied Biosystems Sales Representative.fax, e-mail, or online, and send yoursequences to our synthesizers electroni-cally, speeding order delivery time. Probe Type (3 Quencher) 5 Reporter Dyes Probe Lengths TaqMan® Custom MGB Probes FAM™ VIC® TET™, or NED™* , , 13-25 basesChoice of Quenchers TaqMan® Custom TAMRA™ Dye Probes FAM™ VIC® or TET™ , , Up to 35 basesApplied Biosystems Custom TaqMan®Probes incorporate a 5 reporter dye Table 4.and a 3 quencher. Our most popular *Please note that NED™ dye can give lower signal intensity than FAM™, VIC®, or TET™ dye on most Real-Time PCR systems. The Applied Biosystems 7500 Real-Time PCR System has been optimized to yield higher signal intensity for NED™ dye.quencher is a non-fluorescent quencher(NFQ) combined with an MGB (minorgroove binder) moiety. The NFQ offers Custom TaqMan® Reporter Probes and Primers Part Number Dye Label Quantitythe advantage of lower background TaqMan® TAMRA™ Probes 450025 6-FAM™, VIC® or TET™ 6,000 pmolsignal, which results in better precision 450024 6-FAM , VIC or TET ™ ® ™ 20,000 pmolin quantitation. The MGB moiety 450003 6-FAM™, VIC® or TET™ 50,000 pmolstabilizes the hybridized probe andeffectively raises the melting temperature TaqMan MGB Probes ® 4316034 6-FAM , VIC , NED or TET ™ ® ™ ™ 6,000 pmol(Tm). This means that MGB probes 4316033 6-FAM , VIC , NED or TET ™ ® ™ ™ 20,000 pmolcan be shorter than traditional dual- 4316032 6-FAM™, VIC®, NED™ or TET™ 50,000 pmollabeled probes, which make them better Real-Time PCR Primers 4304970 No label 10,000 pmol (sequence detection primers)suited for allelic discrimination applica- 4304971 No label 80,000 pmoltions. The shorter probe lengths mean 4304972 No label 130,000 pmolthat single base mismatches (e.g., SNPs) Table 9 |
  • 11. TaqMan® MicroRNA Assays• Highly specific—quantitate only mature miRNAs, not inactive precursors A B• Fast, simple, and scalable—two-step assay provides high-quality results in less than three hours• Sensitive—conserves limited samples by requiring only 1–10 nanograms of total RNA or equivalent• Wide dynamic range—up to seven logsBy making novel adaptations in Unparalleled dynamic range. Amplification plot of synthetic miRNA lin-4 by stem-loop RT-PCR. Estimated syntheticassay design, Applied Biosystems is now miRNA input is based on OD: 70, 700, 7,000, 70,000, 700,000, 7M, and 70M copies in PCR. Significant correlationable to bring the specificity, sensitivity, between the copy number and CT value up to seven orders of magnitude. A) amplification plot, B) amplification plot converted to log scale.and simplicity of TaqMan® assays andReal-Time PCR to miRNA detectionand quantitation. conventional design of a random- Requires Only Minimal primed RT step followed by a specific Starting MaterialThe basis of TaqMan® MicroRNA real-time assay. The stem-loop accom- TaqMan miRNA assays are extremelyAssays is a target-specific stem-loop, plishes two goals: 1) specificity for sensitive—researchers need only 1–10reverse-transcriptase primer. Its only the mature miRNA target, and nanograms of purified total RNA orinnovative design (below) overcomes 2) formation of a RT primer/mature equivalent to reliably quantify theira fundamental problem in miRNA miRNA-chimera, extending the 5 end miRNAs of interest, not the severalquantitation: the short length of of the miRNA. The resulting longer RT micrograms typically required formature miRNAs (~22 nt) prohibits amplicon presents a template amenable hybridization-based methods. Such to Real-Time TaqMan assay design sensitivity enables researchers to with great specificity. conduct more extensive experiments looped RT primer with limited starting material, as with mature microRNA To ensure accurate results, every full-panel miRNA expression profiling. individual TaqMan MicroRNA Assay It also conserves researcher’s precious design has been functionally validated Step 1: or difficult-to-obtain materials, such as Stem-loop under laboratory conditions. RT stem cells or matched tumor/normal Distinguish Between Highly human cancer samples. Homologous Mature miRNAs Unparalleled Dynamic Range TaqMan® miRNA assays are not only Step 2: TaqMan miRNA assays can deliver Real-time specific for mature miRNAs, they can up to seven logs of linear dynamic PCR also successfully distinguish between range, from a few copies, to millions forward primer highly homologous targets, and in some of copies (data above). This means cases, with only a single nucleotide mis- that researchers will get accurate data match. This means that researchers can whether their materials contain few be confident that they are measuring or many molecules of the target Q F reverse primer mature, biologically relevant miRNAs, TaqMan® probe miRNA per cell—an important not precursors, and that closely related factor given the wide range of miRNATaqMan® MicroRNA Assay mechanism. A simple, miRNA variants have been targetedtwo-step mechanism brings the advantages of concentrations across different cells, with very high specificity.Real-Time PCR to miRNA quantification. tissue types, and disease states. | pg. 10
  • 12. This increased accuracy also makes Cost-Effective The kit supplies sufficient materials toTaqMan miRNA assays highly repro- With a cost-per-target comparable perform 20 reactions (20 µL volume)ducible among replicates and when to microarray experiments, TaqMan of each assay—enough for severalperformed by different researchers. MicroRNA Assays are a powerful full-panel miRNA screens. alternative. Not only are they conven-Fast Time-to-Results ient and adaptable to high-throughput Because many mature miRNABy taking advantage of gold-standard protocols, they also offer the superior sequences are identical across closelyTaqMan® reagent-based technology specificity, sensitivity, and dynamic related species, the majority of assayswith universal thermal cycling range of Real-Time PCR. in the Human Panel Kit are alsoconditions, TaqMan miRNA assays valid for mouse and rat targets.are familiar, fast, and easy to set up. Case Study: Comparison A cross-species chart detailing theJust start with your total RNA sample, to Northern Analysis human, mouse, and rat microRNAsand get results in less than three Recent studies showed that northern targeted by each individual assayhours using any Applied Biosystems analysis and TaqMan MicroRNA Assay can be downloaded from the Web atReal-Time PCR System. results correlated when both methods were performed under ideal conditions.A Convenient and Scalable Solution However, TaqMan miRNA assays are a 2. TaqMan® MicroRNA AssayTaqMan miRNA assays are pre- better choice because: ABM 000008 hsa-miR-16designed, functionally validated, and This is an individually available assayavailable off-the-shelf from Applied • They can offer a high level of targeting the mature human miRNABiosystems, making them extremely discrimination between highly miR-16. The assay allows customersconvenient. Spend your valuable time homologous miRNA variants to easily evaluate TaqMan MicroRNAgenerating results, not designing and • They are easy to use and provide Assay technology without having totroubleshooting assays. results within three hours invest in the full human panel kit. • They are highly sensitive, requiring Identified as a candidate positiveThe TaqMan miRNA assay format is minimal starting materials control for human miRNA quantifica-easily amenable to high-throughput tion experiments, miR-16 exhibits ascreening methods. All of the up to 157 • They have a dynamic range of high level of expression across mostindividual miRNA assays and controls up to seven logs tissue types. Also, because the maturecontained in the Human Panel – Early Early Access Product Availability miR-16 sequence is identical betweenAccess Kit fit into two 96-well plates. Applied Biosystems has released the human, mouse, and rat, it is also usefulFor example, a statistically rigorous following two early access miRNA for quantifying the mouse and ratexperiment profiling the entire assay products: miR-16 miRNAs.panel in quadruplicate, includingmultiple control assays, can be run 1. TaqMan® MicroRNA Assays Additional TaqMan MicroRNAin just two 384-well plates. Human Panel – Early Access Kit Assay products will become This product includes 157 individual available in the near future. RegisterThe Human Panel Kit provides miRNA assays and three non-human for product updates on the Web atflexibility—researchers can run the negative control assays. The panel mirna.appliedbiosystems.comentire panel together, or any subset covers the majority of humanof assays they wish. This scalable miRNAs currently identifiedsolution accommodates both large in the Sanger miRNA Registryinitial miRNA panel screens, and ( in-depth experiments. | pg. 11
  • 13. Applied Biosystems 7900HT Fast Real-Time PCR System Applied Biosystems 7500HT Fast Real-Time PCR SystemApplied Biosystems 500–660 nm, provides unmatched dye • Relative Quantitation Software allowsReal-Time PCR Systems resolution capabilities gene expression data from up to tenApplied Biosystems Real-Time PCR • Automation Accessory provides walk plates to be combined in one analysisSystems make real-time PCR more away automation for unmatchedaccessible than ever before by providing Applied Biosystems 7300 Real-Time throughput PCR System—an economical solutionpowerful solutions to fit the needs ofany laboratory. These systems are easy to • Enterprise Edition Software enables setting the standard for the basicuse with next generation software, and hundreds to thousands of plates to be researcherof course, they’re backed by Applied analyzed simultaneously and assists • Four color detection providesBiosystems unmatched track record of with 21CFR part 11 compliance the flexibility to perform allperformance, quality, and long-term major applications Applied Biosystems 7500 Fast andreliability. 7500 Real-Time PCR Systems— • Powerful and versatile software versatile platforms for users requiring makes experimental set-up and dataApplied Biosystems 7900HT Fast processing simple and straightforwardReal-Time PCR System—the ultimate extended capabilitiesin performance and flexibility • Advanced five color optical config- • Precision optics and a charge-• User-interchangeable block options uration supports a broader range of coupled device (CCD) camera include 384-well, 96-well, TaqMan® fluorophores, with variable excitation provide highly accurate, Low Density Array, and Fast 96-well capability allowing greater sensitivity reproducible, and reliable results (for results in about 35 minutes) for longer wavelength (red) dyes • Patented sample temperature control• Extended-life 488 nm argon-ion • A new high-speed 96-well thermal provides superior reproducibility and laser combined with continuous cycling block enables real-time PCR consistent, high-quality results wavelength detection from results in under 40 minutespg. 12 |
  • 14. Product Specification Comparison Available Number of Reporter Universal Approximate Part Number Fill Volumes 20 µL Reactions Dye Labels Formulation Delivery TimeTaqMan® Gene Expression Assays Inventoried 4331182 250 µL, 20X 250 FAM™ Yes 2 – 3 days Non-Inventoried 4351372 360 µL, 20X 360 FAM ™ Yes 10 – 14 daysCustom TaqMan® Gene 4331348 360 µL, 20X 360 FAM™ Yes 10 – 14 daysExpression Assays 4332078 750 µL, 20X 750 FAM ™ Yes 10 – 14 days 4332079 967 µL, 60X 2,900 FAM™ Yes 10 – 14 daysTaqMan Endogenous Controls ® Primer limited Various — see Table 1 VIC® Yes Not primer limited Various — see Table 1 FAM™ YesCustom TaqMan Probes ® Various — see Table 5 FAM ™ No 4 – 7 days Various — see Table 5 VIC® No 4 – 7 days Various — see Table 5 TET ™ No 4 – 7 days Various — see Table 5 NED ™ No 4 – 7 days Available Fill Volumes Number of Reporter Universal Approximate Part Number (each assay) Reactions Dye Labels Formulation Delivery TimeTaqMan® MicroRNA Assays(Early Access Products) Human Panel Kit 4365409 RT: 60 µL, 5X; Up to 3,680 FAM™ Yes 2 – 3 days (Includes 157 human 15 µL rxn vol. • 157 individual miRNA assays, and miRNA assays additional non-human (20 rxn each) control assays, for a total TaqMan® Assay: • Additional of up to 3,680 reactions) 40 µL, 10X; negative 20 µL rxn vol. control assays (20 rxn each) Individual hsa-miR-16 assay 4365746 RT: 450 µL, 5X 150 FAM™ Yes 2 – 3 days (Targets human, mouse, 15 µL rxn vol. and rat miR-16) TaqMan® Assay: 300 µL, 10X; 20 µL rxn vol. Reaction Number of Available Approximate Part Number Size 1 µL Reactions Assays Delivery TimeCustom TaqMan® Low Density Arrays Various — see Table 2 1 µL 384 Any Inventoried TaqMan 6 – 8 weeks Gene Expression AssayTaqMan® Low Density Array Panels Various — see Table 2 1 µL 384 Defined Panels of TaqMan 2 – 3 days Gene Expression AssaysTable 6.For Research Use Only. Not for use in diagnostic procedures.NOTICE TO PURCHASER:PLEASE REFER TO THE USER’S GUIDE, PRODUCT INSERT, OR APPLIED BIOSYSTEMS’ PRODUCT PAGE FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION.ABI PRISM, Applied Biosystems, Primer Express, and VIC are registered trademarks and AB (Design), Applera, Celera, FAM, NED, TAMRA, and TET are trademarks of AppleraCorporation or its subsidiaries in the US and/or certain other countries.TaqMan and AmpErase are registered trademarks of Roche Molecular Systems, Inc.©2005 Applied Biosystems. All rights reserved.Printed in the USA, 09/05, Publication 127GU01-03 Headquarters International Sales 850 Lincoln Centre Drive | Foster City, CA 94404 USA For our office locations please call the division Phone 650.638.5800 | Toll Free 800.345.5224 headquarters or refer to our Web site at