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    ZymoScript One-Step RT-qPCR Kit


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    Cat # Name Size
    R3014 ZymoScript One-Step RT-qPCR Kit 100 reactions

    Highlights

    • Fast: Complete cDNA synthesis and robust amplification in as little as 1.5 hours
    • Flexible: Compatible with both SYBR-Green and TaqMan Probe-based assays
    • Efficient: Superior performance compared to competitor RT-qPCR kits
    Description & Documents Specifications FAQ

    Documents

    Supplemental Info

    Product Description

    ZymoScript One-Step RT-qPCR Kit contains all the necessary components for fast and robust RNA quantification in a single kit. Room-temperature reaction setup is quick and easy. The ZymoScript One-Step RT-qPCR Kit eliminates the need to transfer tubes between reverse transcription and qPCR, which minimizes sample handling and reduces the risk of contamination.

    The included Reaction Buffer (4X) contains dNTPs, MgCl2, and other additives to ensure complete cDNA synthesis and specific qPCR amplification in just 1.5 hours. Non-interfering blue tracking dye is included to simplify reaction setup and prevent pipetting errors. The 20X Enzyme Mix containing temperature-activated reverse transcriptase, hot-start DNA polymerase, and RNase inhibitors ensures maximal specificity. The ZymoScript One-Step RT-qPCR kit is compatible with both SYBR-Green and TaqMan probe-based assays for ultimate flexibility. A 20X Fluor Dye for SYBR-Green based detection methods is included for added convenience. Detection of multiple targets in a single reaction (multiplex RT-qPCR) is feasible using TaqMan probes (not provided).

    The ZymoScript One-Step RT-qPCR Kit has been rigorously tested to demonstrate superior sensitivity, specificity, and reproducibility. The kit can reliably quantify as little as 0.1 pg RNA (including difficult GC-rich targets) and outperforms other commercial kits. This kit was also used to develop a molecular diagnostic test for COVID-19 ranked third in sensitivity by the American FDA. The unique buffer system and temperature-activated enzymes facilitate the melting of secondary structures and ensure ultimate specificity for the intended product. ZymoScript One-Step RT-qPCR Kit is the product of choice for simple and sensitive RNA quantification. It is the ideal product to use as a building block for RT-qPCR multiplex assays or high-throughput diagnostic applications. For quick and efficient cDNA synthesis and 2-step RT-qPCR options, see ZymoScript RT PreMix Kit (R3012) and ZymoTaq qPCR Premix (E2054).

    Zymo Research is ready to support your RNA studies and help simplify your RNA quantification workflow. Please contact tech@zymoresearch.com for more information.

    Documents

    Technical Specifications

    Amplicon Length This product performs best with amplicons between 50 bp and 1.5 kb
    Equipment thermal cycler/qPCR instrument
    Ideal Uses high-throughput applications, multiplex diagnostic assays based on RNA targets, routine quantification of specific targets in multiple samples
    Processing Time as little as 1.5 hours
    Reagents Complete and ready-to-use Reaction Buffer (4X) and Enzyme Mix (20X). Fluor Dye (20X) included for SYBR-Green based detection. MgCl2 included to supplement the magnesium concentration if needed.
    Sample Source 0.1 pg - 5µg RNA input
    Size 100 reactions
    Storage Store at -20 °C. Minimize exposure to light.
    Supplemental Info

    Resources

    The amount of total RNA required may vary depending on the expression level of the target transcripts. In general, we recommend using 0.1 pg - 5 µg of input RNA.

    The dye used in the ZymoScript One-Step RT-qPCR Kit has been thoroughly tested in downstream analysis. At the provided concentration, it will not affect fluorescent signal readings during qPCR.

    Lower signal can be expected with degraded and impure RNA samples. We recommend using Quick-RNA or Direct-zol Kits (see Related Products) for high quality RNA extraction.

    A possible reason for missing or delayed amplification is inefficient primer binding to the template. Make sure to use an annealing temperature (step 4 in the protocol provided) at least 2°C below the lowest melting temperature of the primers to ensure efficient binding to the target sequence.

    Yes, the reaction can be assembled at room temperature. The enzymes are only activated at higher temperatures, ensuring maximum specificity.

    We do not recommend storing the kit or kit components at room temperature for prolonged periods of time. The kit can be stored at 4°C for up to 1 week.

    Samples can be treated with DNase I to eliminate DNA contamination. DNase I is included in the Quick-RNA or Direct-zol Kits recommended for RNA extraction. Alternatively, the DNase I set (see Related Products) can be purchased separately.

    No, RNase inhibitors are already included in the enzyme mix to prevent unwanted RNA degradation during reverse transcription.

    For single target detection, SYBR Green based assays offer a more affordable solution. For SYBR Green based assays, add 1 µl of Fluor Dye solution (20X) to each 20 µl RT-qPCR reaction. TaqMan probe-based assays are in general more expensive than SYBR Green based assays. However, they present an advantage when multiple targets are analyzed simultaneously and when a superior level of specificity is required. Do not add Fluor Dye solution to the RT-qPCR reaction when performing TaqMan probe-based assays.

    In general, any commonly used fluorophore (including HEX, VIC, Cy3, Texas Red, Cal Red 610, Quasar 670, Cy5, Cy5.5, and Quasar 705) is compatible with this kit with the exception of FAM fluorophore, which is unstable in our buffer system. Instead, we recommend using Alexa Fluor™ 488, which has overlapping excitation and emission spectra with FAM, but greater stability.

    The Fluor Dye has the same excitation and emission properties as SYBR Green. Activate the SYBR/FAM channel for detection when using the Fluor Dye.

    No, a passive reference dye is not included.

    This depends on different factors, including RNA integrity and primer efficiencies. Generally, this product performs the best with amplicons with a length between 50 bp and 1.5 Kb.

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