KAPA SYBR® FAST One-Step qRT-PCR Kits

Containing the first DNA polymerase evolved for real-time PCR.

KAPA SYBR® FAST One-Step qRT-PCR Kits contain M-MuLV Reverse Transcriptase, RNase Inhibitor and a novel DNA Polymerase engineered via molecular evolution. The kit is optimized for rapid one-step, one-tube RNA quantification. Reduce experimental variation and contamination with a convenient one-step qRT-PCR protocol.

KAPA SYBR® FAST One-Step qRT-PCR Kits are designed to perform optimally in stringent qRT-PCR reaction conditions, exhibiting dramatic improvements in sensitivity, specificity, reaction efficiency, and fluorescence.

Kits are ideally suited for:

  • Gene expression analysis
  • Microarray validation
  • Gene knockdown validation
  • RNAi and miRNA research

Product Description

The KAPA SYBR® FAST One-Step qRT-PCR Kit is a sensitive and convenient solution for real-time PCR using RNA as template. The kit is composed of KAPA SYBR® FAST Master Mix (2X), KAPA RT Mix (50X) and dUTP (10 mM). The KAPA RT Mix (50X) is comprised of wild-type M-MuLV Reverse Transcriptase and RNase Inhibitor and is optimized for rapid one-step, one-tube RNA quantification.

The KAPA SYBR® FAST Master Mix (2X) contains a novel DNA polymerase engineered via molecular evolution and optimized for qPCR using SYBR® Green I dye chemistry. The 2X Master Mix is a ready-to-use cocktail containing all components except primers and template for the amplification and detection of cDNA. KAPA SYBR® FAST One-Step qRT-PCR Universal kits contain ROX reference dye supplied separately. KAPA SYBR® FAST One-Step qRT-PCR ABI Prism kits contain 2X Master Mix with ROX reference dye included in the mix. KAPA SYBR® FAST One-Step qRT-PCR Bio-Rad iCycler Kits contain 2X Master Mix with fluorescein reference dye included in the mix. 

dUTP (10 mM) is also supplied in the kit. The use of dUTP at the recommended final concentration, results in amplicons that can be degraded using Uracil-DNA Glycosylase (UDG). UDG treatment is performed in subsequent reactions in order to minimize carry over PCR contamination downstream. Use of dUTP in this system is optional. UDG is not supplied in this kit.

Product Applications

KAPA SYBR® FAST One-Step qRT-PCR Kits are ideally suited for:

  • Gene expression analysis
  • Low copy gene detection
  • Microarray validation
  • Gene knockdown validation
  • RNAi and miRNA research

Product Performance

Superior signal and reaction efficiency

The improved speed, processivity and robustness of KAPA SYBR® FAST One-Step qRT-PCR kits results in consistently high amplification efficiencies required for accurate relative quantification. To demonstrate the high performance of KAPA SYBR® FAST One-Step qRT-PCR Kit for gene expression analysis, the kit was compared to leading competitor kits across a wide range of input RNA concentrations. The KAPA SYBR® FAST One-Step qRT-PCR Kit achieved higher fluorescence, earlier Ct values and improved amplification efficiency when compared to competitor one-step qRT-PCR kits.

Higher fluorescence, earlier Ct values and improved reaction efficiency. The RRMI gene (94 bp, 45.7% GC) was amplified from log-fold serial dilutions of RNA (100 ng- 10 pg/20 μl reaction), isolated from human placenta, using the KAPA SYBR® FAST One-Step qRT-PCR Kit (green) and Competitor I (blue). Linear amplification plots demonstrate earlier Ct values and greater baseline subtracted fluorescence for the RRMI gene with the KAPA SYBR® FAST One-Step qRT-PCR Kit. Calculated reaction efficiencies confirmed consistently high performance is achievable with the KAPA SYBR® FAST One-Step qRT-PCR Kit (RRMI = 100% and 97%). Efficiencies obtained with Competitor I (105%) were sub-optimal. Reactions were performed using the competitors recommend protocols on the Eppendorf® MasterCycler™ ep realplex4 S real-time PCR cycler.

Higher fluorescence, earlier Ct values and improved reaction efficiency. The RRMI gene (94 bp, 45.7% GC) was amplified from log-fold serial dilutions of RNA (100 ng- 10 pg/20 μl reaction), isolated from human placenta, using the KAPA SYBR® FAST One-Step qRT-PCR Kit (green) and Competitor Q (purple). Linear amplification plots demonstrate earlier Ct values and greater baseline subtracted fluorescence for the RRMI gene with the KAPA SYBR® FAST One-Step qRT-PCR Kit. Calculated reaction efficiencies confirmed consistently high performance is achievable with the KAPA SYBR® FAST One-Step qRT-PCR Kit (RRMI = 100% and 97%). Efficiencies obtained with Competitor Q (118%) were sub-optimal. Reactions were performed using the competitors recommend protocols on the Eppendorf® MasterCycler™ ep realplex4 S real-time PCR cycler.

Higher fluorescence, earlier Ct values and improved reaction efficiency. The RRMI gene (94 bp, 45.7% GC) was amplified from log-fold serial dilutions of RNA (100 ng- 10 pg/20 μl reaction), isolated from human placenta, using the KAPA SYBR® FAST One-Step qRT-PCR Kit (green) and Competitor A (orange). Linear amplification plots demonstrate earlier Ct values and greater baseline subtracted fluorescence for the RRMI gene with the KAPA SYBR® FAST One-Step qRT-PCR Kit. Calculated reaction efficiencies confirmed consistently high performance is achievable with the KAPA SYBR® FAST One-Step qRT-PCR Kit (RRMI = 100% and 97%). Efficiencies obtained with Competitor A (176%) were sub-optimal. Reactions were performed using the competitors recommend protocols on the Eppendorf® MasterCycler™ ep realplex4 S real-time PCR cycler.

Robust amplification across a broad range of target lengths

To demonstrate the robust performance of the KAPA SYBR® FAST One-Step qRT-PCR Kit, MS2 bacteriophage RNA targets of increasing length were amplified at a concentration of 0.8 pg/20 μl using the KAPA SYBR® FAST One-Step qRT-PCR Kit (green) or Competitor T (red), Competitor I (blue), and Competitor Q (purple). Increased amplicon length led to a significant decrease in competitor performance as indicated by delayed Ct values or complete reaction failure. cDNA synthesis and qPCR amplification were performed using the competitors recommend protocols on the Eppendorf® MasterCycler™ ep realplex4 S real-time PCR cycler.

High reproducibility and sensitivity across a broad range of concentrations

The high reproducibility and sensitivity of the KAPA SYBR® FAST One-Step qRT-PCR Kit enable accurate interrogation of a broad range of RNA template concentrations. A 279 bp fragment was amplified in replicates of six from log-fold serial dilutions of commercial MS2 bacteriophage RNA (80 pg - 0.8 fg/20 μl). cDNA synthesis was performed at 42 oC for 5 min followed by 95 ºC for 5 min and 40 cycles of 3 sec denaturation at 95 ºC and 30 sec combined annealing/extension at 60 ºC. The calculated reaction efficiency of 95% was obtained using all six replicates per data point. Reactions were performed using the competitors recommend protocols on the Eppendorf® MasterCycler™ ep realplex4 S real-time PCR cycler.

Storage and handling

Upon arrival, store kit components away from light and at –20 °C in a constant-temperature freezer. When stored under these conditions and handled correctly, full activity of the master mix is retained for 18 months from the date of receipt. The KAPA RT Mix (50X) is guaranteed for 6 months at -20 °C.

Minimize exposure of the Master Mix (2X) to direct light. Exposure to direct light for an extended period of time may result in loss of fluorescent signal intensity. Always ensure that the product has been fully thawed and mixed before use.

License Information

Certain applications of this product are covered by patents issued to parties other than Kapa Biosystems and applicable in certain countries. Purchase of this product does not include a license to perform any such applications. Users of this product may therefore be required to obtain a patent license depending upon the particular application and country in which the product is used.

Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,994,056, 6,171,785, and 5,928,907 (claim numbers 12-24, 27-28). The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim (such as apparatus or system claims in US Patent No. 6,814,934) and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only.  Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.

This product is provided under an agreement between Molecular Probes, Inc. and Kapa Biosystems Inc., and the manufacture, use, sale or import of this product is subject to one or more of U.S. Patent Nos. 5,436,134; 5,658,751 and corresponding international equivalents, owned by Molecular Probes, Inc. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer, where such research does not include testing, analysis or screening services for any third party in return for compensation on a per test basis. The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this product for purposes other than research, contact Molecular Probes, Inc., Business Development, 29851.

Licensed under U.S. Patent nos. 5,338,671 and 5,587,287 and corresponding patents in other countries.

The purchase of this product includes a limited, non-transferable license under specific claims of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, owned by the University of Utah Research Foundation or Evotec Biosystems GmbH and licensed to Idaho Technology, Inc. and Roche Diagnostics GmbH, to use only the enclosed amount of product according to the specified protocols.  No right is conveyed, expressly, by implication, or by estoppel, to use any instrument or system under any claim of U.S. Patent Nos. 6,174,670, 6,569,627 and 5,871,908, other than for the amount of product contained herein.

SYBR® is a registered trademark of Molecular Probes, Inc, Oregon. PRISM® and GeneAmp® are registered trademarks of Applera Corporation. iCycler®, Mx3000P®, Mx3005™, Mx4000®, Roto-Gene™, DNA Engine Opticon™, Chromo 4™, LightCycler® , and Smart Cycler® are trademarks or registered trademarks of their respective companies.