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ZymoScript One-Step RT-qPCR Kit
ZymoScript One-Step RT-qPCR Kit
- 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
ZymoScript One-Step RT-qPCR Kit contains all the necessary components for fast reverse transcription and robust qPCR amplification 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 RT-qPCR. 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 workflow. Please contact email@example.com for more information.
|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|
|Storage||Store at -20 °C. Minimize exposure to light.|
Q1: What is the minimum/maximum amount of RNA template I should use?
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.
Q2: Will the blue tracking dye interfere with qPCR fluorophore channels and affect the signal of probe/fluorescent dyes?
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.
Q3: How is the performance of the ZymoScript One-Step RT-qPCR kit with low-quality RNA samples?
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.
Q4: RT-qPCR signal is not detected, or it is detected at higher Ct than expected. What should I do?
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.
Q5: Can I assemble the RT reaction at room temperature?
Yes, the reaction can be assembled at room temperature. The enzymes are only activated at higher temperatures, ensuring maximum specificity.
Q6: Can I store the ZymoScript One-Step RT-qPCR kit at Room Temperature or 4°C?
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.
Q7: How can I ensure that there is no genomic DNA contamination in my RT reaction?
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.
Q8: Do I need to add RNase inhibitors?
No, RNase inhibitors are already included in the enzyme mix to prevent unwanted RNA degradation during reverse transcription.
Q9: Should I use SYBR Green or a TaqMan probe-based assay?
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.
Q10: What fluorophores are recommended for 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.
Q11: Which channel should be activated when using the Fluor Dye solution?
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.
Q12: Does ZymoScript One-Step RT-qPCR contain a passive reference dye?
No, a passive reference dye is not included.
Q13: What is the amplicon length that can be achieved using the ZymoScript One-Step RT-qPCR Kit?
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.