ZipScript and ZipScript WarmX One-Step RT-qPCR Kits

• Highly sensitive, specific and reproducible RT-qPCR solution
• Efficient multiplex one-step RT-qPCR formulation
• ZipScript features an optimized blend of EnzScript RT and Phoenix Hot Start DNA Polymerase
• ZipScript WarmX is formulated with an aptamer-based RTase
• ZipScript WarmX reduces non-specific reverse transcription during reaction setup
• ZipScript WarmX is automation friendly for room temperature setup

The ZipScript One-Step RT-qPCR Kit and ZipScript WarmX One-Step RT-qPCR Kit are highly sensitive and reproducible RT-qPCR solutions optimized for real-time PCR. The high-efficiency RNA-dependent multiplex qPCR formulation enables a broad range of sample detection. ZipScript features a blend of RNase H minus reverse transcriptase (EnzScript Reverse Transcriptase) and a fast-activating hot start DNA polymerase (Phoenix Hot Start Taq DNA Polymerase). For operational convenience, detect as little as 0.1 pg of RNA in a four plex assay with the one-step reaction format. In addition to market-leading RT-qPCR performance, world-class manufacturing by QIAGEN ensures consistent, repeatable results in your laboratory.

The ZipScript WarmX One-Step RT-qPCR Kit features an aptamer based warm-start capacity that blocks reverse transcription during reaction setup at room temperature, improving assay specificity and consistency. Reactions can be set up at room temperature with more confidence, especially when using high-throughput automated solutions.

Supplied with: 
2X ZipScript Reaction Buffer 1 (B7641L)
Inquire for lyophilized or lyo-ready options.

ZipScript One-Step RT-qPCR Mix is a blend of EnzScript Reverse Transcriptase and Phoenix Hot Start Taq DNA Polymerase enzymes. EnzScript is a robust, RNA-dependent DNA polymerase with no detectable RNase H activity. The enzyme can be used to generate first-strand cDNA from mRNA or total RNA within an optimal temperature range between 42–50ºC. 

The Phoenix Polymerase is a recombinant, thermostable Taq DNA polymerase complexed with a thermolabile, neutralizing antibody. This fast-activating antibody-mediated hot-start capability enhances the overall specificity, sensitivity, and yield of the PCR reaction by reducing nonspecific amplification and primer-dimer formation prior to cycling. Together with EnzScript, Phoenix creates ZipScript, an extremely sensitive RT-qPCR mix with: 

• Broad detection range: High sensitivity (<0.1pg total human RNA) combined with a broad range of detection (>7 orders of 
  magnitude).
• Superior multiplexing: Multiplex up to 4 targets per reaction.
• Market-leading performance: Superior sensitivity, specificity, reproducibility and efficiency compared to existing industry-leading formulations.
• Reliable throughput: Ideal for high-throughput environments where ease of use, automation compatibility, and result 
  accuracy are critical.
• Backed by quality: QIAGEN quality ensures lot-to-lot consistency, reducing inter-assay variability and maximizing data 
  validity.

The aptamer based warm-start reverse transcriptase in the 10X ZipScript WarmX One-Step RT-qPCR Mix reduces non-specific reverse transcription during reaction setup at room temperature and improves assay specificity and consistency. The addition of the WarmX feature facilitates high-throughput automation solutions. 

ZipScript WarmX:

• Reduces non-specific and spurious reverse transcription during reaction setup at room temperature.
• Maintains robust assay performance after extended (up to 24 hours) preincubation at room temperature.
• Prevents loss of detection or lower amplitude resulting from room temperature preincubation.

Mix properties

• Storage temperature: –25°C to –15°C

ZipScript One-Step RT-qPCR Mix is a blend of EnzScript Reverse Transcriptase and Phoenix Hot Start Taq DNA Polymerase enzymes.

Reverse transcriptases polymerize a strand of DNA (cDNA) that is complimentary to an original RNA template. The cDNA can then be further amplified through PCR, and qPCR for downstream analysis of transcripts and gene expression. Native retroviral RT has three sequential biochemical activities: RNA-dependent DNA polymerase activity, ribonuclease H (RNase H) activity and DNA-dependent DNA polymerase activity. Collectively, these activities enable the enzyme to convert single-stranded RNA into double-stranded cDNA. RNase H activity degrades the RNA strand of a DNA/RNA hybrid and frees the newly made DNA strand for use as a template in the synthesis of the second strand of DNA. RNase H activity is considered disadvantageous for synthesis of long cDNAs due to the potential of the enzyme to degrade the RNA template before completion of full-length reverse transcription. 

EnzScript Reverse Transcriptase has no detectable RNase H activity and has increased thermostability. The enzyme is suitable for generating first-strand cDNA from poly(A) mRNA or total RNA for use in downstream applications such as RT-PCR, cDNA cloning or library construction for RNA-Seq.

Phoenix Hot Start Taq DNA Polymerase is a recombinant, thermostable Taq DNA polymerase complexed with a thermostable, neutralizing antibody that blocks the 5ʹ→3ʹ polymerase activity prior to the initial DNA denaturation step of PCR. Such antibody-mediated hot-start capability enhances the overall specificity, sensitivity and yield of the PCR by reducing nonspecific amplification and primer–dimer formation prior to PCR cycling and allows the convenience of reaction set up at room temperature. 

To improve assay specificity and consistency especially for high-throughput automated solutions, the ZipScript WarmX One-Step RT-qPCR Kit features an aptamer-based warm-start RTase that blocks reverse transcription during reaction setup at room temperature. The two-phase warm/hot-start procedure ensures robust reaction performance, allowing extensive reaction setup at room temperature with confidence.

ZipScript One-Step RT-qPCR reaction setup

1. All components and reaction set up should be kept on ice.
2. Thaw the 2x ZipScript Reaction Buffer I completely and vortex for 3–5 seconds to mix thoroughly. Quick spin to collect contents if necessary.
3. Prepare primer and probe mixture. A final concentration of 0.4–0.9 µM for each primer and 0.1–0.5 µM for probe is recommended. However, the optimal concentration for primers and probe needs to be empirically determined for each assay.
4. Determine the number of reactions to prepare, including No Template Controls (NTCs). Add 10% extra volume to compensate for the pipetting loss.
5. Use the amounts listed in the table below to set up the reaction mix. We recommend making a master mix to minimize variations and potential errors.

   Components	Volume per reaction	Final concentration
   Nuclease-free water	To a final reaction volume of 20 µL	–
   2x ZipScript Reaction Buffer I	10 µL	1X
   50x ROX (optional)	0.4 µL	1X
   Primer and probe mixture	X µL	Variable
   25x ZipScript Enzyme Mix	0.8 µL	1X
   RNA Template	Variable	Variable
6. Seal PCR plate and spin briefly to bring down reagents.
7. Program the cycling conditions based on the recommendations below.

   Steps	Temperature	Time	Cycles
   Reverse transcription	50°C	15 minutes	1
   Taq activation and initial denaturation	95°C	2 minutes	1
   Denaturation	95°C	15 seconds	40
   Annealing and extension*	60°C	30–60 seconds

   * Cycling parameters can be modified (especially annealing and extension parameters) to fit specific primer and probe selection.

   ZipScript WarmX One-Step RT-qPCR Reaction Setup

             1. Thaw the 2X ZipScript Reaction Buffer I completely and vortex for 3–5 seconds to mix thoroughly. A quick spin to collect contents if necessary.

             2. Prepare primer and probe mixture. A final concentration of 0.4–0.9 µM for each primer and 0.1–0.5 µM for probe is recommended. However, the optimal concentration for primers and probes needs to be empirically determined for each assay.

             3. Determine the number of reactions to prepare, including No Template Controls (NTCs). Add 10% extra volume to compensate for the pipetting loss.

             4. Use the amounts listed in the table below to set up the reaction mix. We recommend making a master mix to minimize variations and potential errors.

             5. Seal the PCR plate and spin briefly to bring down the reagents.

             6. Program the cycling conditions based on the recommendations below.

* Cycling parameters can be modified (especially annealing and extension parameters) to fit specific primer and probe selection.

Quality control analysis

The functionality of the ZipScript is evaluated by amplification of three mRNA transcripts in a one-step RT-qPCR assay. The amplification threshold (Cq) of the test lot is compared to a reference lot.

The functionality of the ZipScript WarmX is evaluated by amplification of three mRNA transcripts in a one-step RT-qPCR assay. The amplification threshold (Cq) of the test lot is compared to a reference lot.

Warm-start Function Assay: The warm-start feature of ZipScript WarmX is tested by SYBR-based RT-qPCR amplification of an mRNA transcript after a 24-hour pre-incubation of the reaction at 25°C. Melting curve analysis confirmed that no non-specific amplification is detected.

Enzyme components were tested prior to formulation of the master mix and found free of contaminating endonucleases and exonucleases. Enzyme purity was >99% as determined by SDS-PAGE, and negligible E. coli genomic DNA contamination was confirmed by qPCR. Specific activity was verified for each enzyme pre-formulation.

This product is available for molecular biology applications such as:

• One-step RT-qPCR
• Gene expression analysis of RNA targets
• DNA and RNA detection and quantification
• ZipScript WarmX is ideal for one-step RT-qPCR applications with high throughput automation solutions