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.

Frequently Asked Questions

1. What is the difference between a one-step and a two-step real-time PCR reaction?

In a one-step qRT-PCR reaction, reverse transcription and quantitative PCR are performed as a single, closed tube assay, reducing the risk of contamination. Fewer pipetting steps and ease-of-use make one-step qRT-PCR ideal for routine, high-throughput screening. In two-step qRT-PCR reactions, reverse transcription and quantitative PCR are performed as separate reactions in separate tubes. Two-step qRT-PCR provides more flexibility since cDNA can be archived and used for additional real-time PCR reactions, eliminating the need to continually isolate RNA. However this flexibility comes with an increased risk contamination and prolonged set-up time.

2. What testing should be performed in order to assess the quality of an RNA sample?

All isolated RNA can be assessed electrophoretically on a denaturing 2% agarose gel containing ethidum bromide. The gel should contain sharp 28S and 18S rRNA bands with no smearing at their low molecular weight edge.  If utilizing the Agilent BioAnalyzer, the RNA Integrity Number (RIN) should be above 5.0.

3. What type of reverse transcriptase is present in the 50X KAPA RT Mix?

The 50X KAPA RT Mix contains wild-type M-MuLV Reverse Transcriptase that allows cDNA synthesis to occur at low temperatures. In addition, an RNase Inhibitor is also included in this enzyme mix to prevent RNA degradation due to RNase contamination.

4. What template is compatible with the kit? 

KAPA SYBR FAST One-Step qRT-PCR kits can be used with most RNA as template e.g. human, mouse, rat and bacteria.

5. How much RNA should be used per reaction?

Suggested input quantities of template for KAPA SYBR FAST One-Step qRT-PCR assays are: 1 pg to 1 µg total RNA,10 fg to 100 ng poly A(+) RNA , 10 to 1x10E8 copies viral RNA.

6. Can  reactions be set-up at room temperature?

No. The reactions must be set-up on ice. The  50X KAPA RT Mix is not thermostable, hence all reaction components must be kept cold during set-up.

7. Why should I treat my RNA with DNase?

RNA should be treated with RNase-free DNase to remove genomic DNA (gDNA) contamination prior to reverse transcription in order to eliminate gDNA as a source of template for qRT-PCR. Ideally, any new RNA sample should be assessed for DNA contamination (use a no-reverse transcription control). This is most important when low copy mRNAs are being interrogated and is dependent on the level of DNA contamination in the sample. For low copy transcripts, the level of DNA contamination can alter quantification results significantly. Alternatively you can design primers that are intron spanning to ensure only cDNA is amplified.

8. Will inactivated DNase affect the quality of the qPCR result?

Yes. Any residual DNase activity has the potential to degrade cDNA product and thereby reduce the amount of template available for qPCR amplification. DNase will not be active during PCR cycling, however, it could degrade primers prior to the cycling process.

9. Why is the dUTP supplied in a separate tube?

dUTP is supplied in a separate tube to offer optional treatment with UDG if prevention of carry-over contamination in subsequent reactions is required. UNG is NOT supplied in KAPA SYBR FAST qRT-PCR Kits.

10. Can I perform UNG decontamination when performing one-step qRT-PCR?

It is not recommended to use UNG when performing reverse transcription. When using a dNTP mix with dUTP in a reverse transcription reaction, uracil will be incorporated into the cDNA generated from your RNA template. The UNG will cleave the DNA containing dUTP residues and result in inefficient or no amplification.

11. What type of priming method can I use with the KAPA SYBR FAST One-Step qRT-PCR Kit?

Only gene-specific primers can be used in a one-step qRT-PCR system. 

12. Can the temperature of the cDNA synthesis step be adjusted?

Optimal cDNA synthesis using wild-type M-MuLV RT occurs at 42 ºC, however in cases of difficult templates, temperature adjustment between 42 ºC - 50 ºC can be performed.

13. When is a 3-step PCR cycling protocol recommended?

If the PCR efficiency is still sub-optimal after adjusting combined annealing/extension times and temperatures or when annealing temperatures of primers are less than 60 ºC, it is advisable to convert to a 3-step PCR protocol.  If, the optimal annealing temperature is 55 ºC, then perform annealing for 20 sec at 55 ºC followed by 1 sec extension and data acquisition at 72º C.

14. Why do certain KAPA SYBR FAST One-Step qRT-PCR kits contain a ROX passive dye?

ROX dye is used to normalize the emission recorded from the reporter dye (SYBR Green I) in later cycles in some instruments. ROX compensates for small fluorescent fluctuations such as bubbles and well-to-well variations that may occur.

15. How do I eliminate primer dimer in the no template control (NTC)? 

The reasons for primer dimer formation in a NTC are often due to multiple factors.  These include: sub-optimal primer annealing temperature (often due to differences in buffering conditions between different qPCR kits), sub-optimal primer synthesis (HPLC purified primers result in less primer dimer formation and are useful for low copy number detection), and poor primer design (using software such as Primer3 is recommended in primer design).  Reducing the total number of cycles in a qPCR reaction is an alternative method if amplification of the primer dimer lies outside of the range of the experimental data.  For example, if the sample being interrogated has a Ct of 28 cycles, and contains specific product only (no primer dimer), and the NTC amplifies a primer dimer after 37 cycles, the reaction can be run for 35 cycles, rather than 40.  This approach can be taken only when the sample being tested gives rise to specific product and not a combination of specific product and primer dimer.

16. How should the KAPA SYBR® FAST One-Step qRT-PCR kit be stored and when does it expire?

All kit components should be stored at -20 ºC. Extra care should be taken when handling the 50X KAPA RT Mix. The RT Mix should always be stored at -20 ºC as the enzymes are not thermostable. The KAPA SYBR® FAST qPCR Master Mix is sensitive to light and should be protected from direct light particularly during storage.  Care should be taken to avoid freeze-thaw cycles. When stored under these conditions and handled correctly, full activity of the kit is retained for at least 12 months as indicated on the kit label.